Use of regulatory sequences for specific, transient expression in neuronal determined cells

ABSTRACT

The present invention relates to the use of regulatory sequences for mediating specific, early transient expression in proliverative neuronal determined cells. Furthermore, the uses of recombinant nucleic acid molecules comprising said defined regulatory sequences for mediating specific, early transient expression in proliverative neuronal determined cells as well as for the generation of non-human transgenic organisms and/or host cells are disclosed. In addition, the invention provides for transgenic non-human animals and/or host cells comprising said regulatory sequences and/or recombinant nucleic acid molecules. The invention also describes methods for the preparation of such vectors, host cells and transgenic nonhuman animals as well as methods for the detection and/or isolation of neuronal determined cells. Additionally, methods for screening of compounds capable of regulating neuronal determined cell activity, neurogenesis, stimulating proliferation of neuronally committed precursor cells and/or neuronal differentiation are provided and the invention also relates to methods for the detection and analysis of neuronal differentiation, neuronal migration and/or neuronal determination processes. Finally, the invention relates to diagnostic and pharmaceutical compositions comprising the regulatory sequences, recombinant nucleic acid molecules, host-cells or isolated neuronal determined cells described herein.

Use of regulatory sequences for specific, transient expression inneuronal The present invention relates to the use of regulatorysequences for mediating specific, early transient expression inproliferative neuronal determined cells. Furthermore, the uses ofrecombinant nucleic acid molecules comprising said defined regulatorysequences for mediating specific, early transient expression inproliferative neuronal determined cells as well as for the generation ofnon-human transgenic organisms and/or host cells are disclosed. Inaddition, the invention provides for transgenic non-human animals and/orhost cells comprising said regulatory sequences and/or recombinantnucleic acid molecules. The invention also describes methods for thepreparation of such vectors, host cells and transgenic non-human animalsas well as methods for the detection and/or isolation of neuronaldetermined cells. Additionally, methods for screening of compoundscapable of regulating neuronal determined cell activity, neurogenesis,stimulating proliferation of neuronally committed precursor cells and/orneuronal differentiation are provided and the invention also relates tomethods for the detection and analysis of neuronal differentiation,neuronal migration and/or neuronal determination processes. Finally, theinvention relates to diagnostic and pharmaceutical compositionscomprising the regulatory sequences, recombinant nucleic acid molecules,host-cells or isolated neuronal determined cells described herein.

Multipotent neural stem cells (NSCs) from the developing and adult brainproliferate, self renew and give rise to neurons, astrocytes andoligodendroglia, the three major cell types of the central nervoussystem (CNS). Neurogenesis, the neuronal differentiation of multipotentNSCs, requires cell fate commitment, neuronal lineage restriction andsubsequent differentiation and maturation. Subsequent changes in cellidentity from NSCs to neuroblasts, neuronal restricted/determinedprecursor cells and mature neurons are regulated by intrinsic changes ingene expression. Multipotent NSCs are characterized by expression of“markers”, like nestin, Notch1 and Musashi (see Lendahl, Cell 60 (1990),585-595). However, these markers are not restricted to NSCs, andtherefore insufficient for the identification of NSCs. For example,Musashi is found in hepatic cells, testis sertoli cells and cord bloodcells, as shown in Shu, Biochem. Biophys. Res. Commun. 293 (2002),150-154; Saunders, Biol. Reprod. 66 (2002), 500-507 and Sanchez-Ramos,Exp. Neurol. 171 (2001), 109-115. Neuronal restricted/determinedprecursor cells are characterized by expression of PSA-NCAM (Doetsch, J.Neurosci. 17 (1997), 5046-5061). However, PSA-NCAM expression is notrestricted to neuronal determined precursor cells, but it is also foundon oligodendrocyte precursors; see Grinspan, J. Neurosci. Res. 41(1995), 540-551. The βIII isotype of tubulin is used as an earlyneuronal marker (inter alia, Palmer, Nature 411 (2001), 4243). Itsexpression, however, is not restricted to neuronal determined precursorcells, but can still be detected in some mature neurons and also innon-neuronal cells from the pigment epithelium as shown by Moskowitz, J.Neurosci. Res. 34 (1993), 129-134 and Vinores, Exp. Eye Res. 60 (1995),385-400.

The adult mammalian CNS, although classically seen as a non-regenerativetissue, retains the potential to generate new neurons. Two areas ofadult neurogenesis are well documented: the dentate gyrus of thehippocampal formation and the lateral ventricle wall/olfactory bulbaxis, as first described by Altman ((1965) J. Comp. Neurol 124, 319-335and (1969) J. Comp. Neurol. 137, 433-457). Within the hippocampus, newlygenerated neurons arise from proliferating cells at the border betweenthe hilus and the granule cell layer, see (Cameron, Neuroscience 56(1993), 337-344; Kuhn, J. Neurosci 16 (1996), 2027-2033 or Seri, J.Neurosci 21 (2001), 7153-7160). Neurogenesis of hippocampal granulecells continues throughout life, although a steady decline is observedwith aging (Kuhn (1996), loc.cit; Kempermann, J. Neuroscience 18 (1998),3206-3212). Cells destined for the olfactory bulb (OB), originate from apopulation of neural stem cells and progenitors dividing in the wall ofthe lateral ventricle (Doetsch, J. Neurosci 17 (1997), 5046-5061). Thenewly generated neuronal restricted/determined precursor cells migratetowards the OB along a structure referred to as the rostral migratorystream (RMS). In the OB, the incoming neuronal restricted precursorcells integrate and complete differentiation as granule cells andperiglomerular neurons (Betarbet, J. Dev. Neurosci 14 (1996), 921-930;Winner, Eur J. Neurosci. 16(9 (2002)),1681-1689).

Although neurogenesis still takes place in the adult mammalian CNS, thedamaged brain is largely incapable of functionally significantstructural self-repair. This reflects the incapacity of NSCs andneuronal restricted precursors present in the neurogenic regions tomigrate toward the damaged regions in large amount and rebuildcircuitries. In order to reach a critical mass of replacement cells in aspecific CNS location, cell grafting appears to be a promising approach.Different sources of cells, such as dissociated fetal mesencephalictissue, in vitro expanded stem cells derived from blastocysts orembryonic forebrains and neural stem cells from the adult brain havebeen investigated for their potential use in transplantationexperiments, see, inter alia, Brundin, Brain 123 (2000), 1380-1390;Freed. N. Engl. J. Med. 344 (2001), 710-719; Bjorklund, Proc. Natl.Acad. Sci. USA 99 (2002), 2344-2349; Brustle, Curr. Opin. Neurobiol. 6(1996), 688-695; Englund, Exp. Neurol. 173 (2002), 1-21 or Gage, Science287 (2000), 1433-1438. In humans suffering of Parkinson's disease,implantation of fetal tissue has resulted in some degree of functionalrecovery (Piccini, Nat. Neurosci. 2 (1999), 1137-1140; Brundin (2000),loc. cit. or Freed (2001), loc.cit.). However, the highly limited accessto fetal tissue and the ethical concerns surrounding its use in patientshave strengthened the search for alternative neuronal-restrictedprecursor cell sources, in particular those harvested from adulttissues. Only recently, first attempts for the direct isolation of NSCsby cell surface antigens or by promoter driven reporter gene expressionwere undertaken. Multipotent NSCs were directly isolated from fetalhuman brain tissue using positive selection for CD133 and negativeselection for CD34 and CD45 (Uchida, Proc. Natl. Acad. Sci. USA 97(2000), 14720-14725) or selection for the extracellular domain of Notch1(Johansson (1999), loc.cit). However, both approaches have theirlimitations, since neither CD133, nor Notch1 expression is specific forneural stem cells, as shown in Yin, Blood 90 (1997), 5002-5012 or Stier,Blood 99 (2002), 2369-2378. Genetic markers such as the neural stem cellspecific enhancer element of the nestin gene, the pan-neuronal tubulinα1 (Tα1) promoter or the oligodendroglia precursor 2′,3′-cyclicnucleotide 3′-phosphodiesterase (CNP) promoter were employed for theisolation and enrichment of specific neuronal progenitor populations byexpressing green fluorescent protein (GFP) under control of thedifferent cell type promoter/enhancer elements; see Roy, J. Neurosci.Res. 59 (2000), 321-331, Wang, Dev. Neurosci. 22 (2000), 167-176; Roy,J. Neurosci. 19 (1999), 9986-9995; Sawamoto, J. Neurosci. Res. 65(2001), 220-227; Kawaguchi, Mol. Cell. Neurosci. 17 (2001)m 259-273; WO00/23571; WO 98/32879 or WO 01/53503. Yet, the genetic elements employedare merely useful for the isolation of certain cell populations: thenestin-promoter element is useful for the isolation of multipotentneural stem cells, the tubulin al (Tα1) promoter for differentiatedneurons, and the CNP promoter for the isolation of oligodendrocyteprecursors. None of the genetic elements described so far is specificfor and can be used for the detection and isolation of neuronalrestricted/determined precursor cells, in particular cells which arecapable of further proliferation. Such a genetic element or such amarker should ideally be induced in the neuronal restricted precursorpopulation, in order to detect the new neurons while being generated,and later downregulated in mature neurons. Progress in the field ofneurogenesis, NSC (neural stem cell) biology, cell isolation techniquesand future clinical applications for NSCs is currently limited by thelack of cell type specific gene or marker expression, in particularmarkers for neuroblasts, neuronal determined cells or neuronal precursorcells which uniquely lead to neurons. There is a clear need for theidentification and introduction of such markers.

Adult neurogenesis is typically detected by incorporation ofbromodeoxyuridine (BrdU) into dividing cells and co-labeling ofBrdU-positive cells with markers for mature neurons. BrdU is abirth-dating marker which permanently labels cells born at the moment ofBrdU application and allows, in combination with cell type specificmarkers, distinct cell fate analysis. Yet, such methods, like theBrdU-incorporation has severe limitations in medical and scientific use,due, inter alia, to the technical and physiological hurdles and thetoxicological properties of BrdU. Furthermore, retroviral labeling hasbeen used to track dividing cells and their cell fate within the CNS.However, these methods have their limitations in the detection ofneurogenesis (Cooper-Kuhn, Brain Res. Dev. Brain. Res. 134 (2002),13-21). For example, retroviral incorporation requires invasiveintracranial injection, which causes parenchymal lesions and possibleinflammatory reactions. Moreover, diffusion barriers prevent retrovirallabeling of larger progenitor populations. Finally, though thetransfection is stable, virally encoded reporter genes may bedownregulated with time (Duch, J. Virol 68 (1994), 5596-5601). On theother hand, BrdU, which integrates in the DNA of dividing cells, isdiluted within the progeny of a labeled cell after multiple subsequentdivisions. The difficulties associated with these methods andincompatibility with human tissue analysis call for new, specific andquantifiable indicators of neurogenesis.

During the development of the central nervous system, the microtubulebinding protein doublecortin (DCX) is associated with migration ofneuroblasts. Besides this developmental role, expression of DCX remainshigh within certain areas of the adult mammalian brain. These areas,mainly the dentate gyrus and the lateral ventricle wall in conjunctionwith the rostral migratory stream and olfactory bulb, retain thecapacity to generate new neurons into adulthood.

Doublecortin (DCX) is first detected at mouse embryonic day 10.5, DCX isexpressed at high levels in the developing mouse CNS (des Portes, Cell92 (1998), 51-61; Francis, Neuron 23 (1999), 247-256 and WO 99/27089).DCX expression is retained within the areas of continuous neurogenesisin the adult brain (Nacher, Eur. J. Neurosci 14 (2001), 629-644). Themorphology of DCX-expressing cells is consistent with that of migratingneurons. Moreover, many of these cells were co-labeled with PSA-NCAM, anantigen also present on migrating neurons (Bonfanti, Neurosci 62 (1994),291-305). The Dcx gene was originally described in the context of humancortical disorders (Gleeson Cell 92 (1998), 63-72). Mutant alleles ofDcx provoke a migratory impairment of neurons and lead to corticaldysplasia (des Portes (1998), loc. cit.; Gleeson (1998) loc. cit.; andCouillard-Despres, Curr. Mol. Med. 1 (2001), 677-688).

As mentioned herein above, some further neuro-system related markers orexpression patterns have been described in the art. For example, WO93/07280 describes astrocyte-specific transcription of human genes. Inparticular, WO 93/07280 discloses sequences capable of regulatingastrocyte-specific transcription of glial fibrillary acidic protein(GFAP). WO 95/25792 describes an endogenous neuron promoter expressedduring growth of both developing and mature neurons, namely the Tα1α-tubulin promoter. According to WO 95/25792 this promoter drivesexpression in embryonic neurons, neurons of newborns and remainsexpressed in adult neurons. In WO 98/32879 a plurality of promoters areemployed in a method for separating cells, wherein some of the describedpromoters are either neuronal or neuron-specific promoters comprisingthe enolase promoter, MAP-1B promoter, decarboxylase promoter, dopamineβ-hydroxylase promoter, NCAM promoter, HES-5, HLH protein promoter, theα1 tubulin promoter (also described in the above-mentioned WO 95/25792),α-internexin promoter, peripherin promoter or the GAP43 promoter. Allthe promoters mentioned in WO 98/32879 are promoters which are notspecific for neuron-specific early progenitor cells. In particular aredisclosed: the neuron-specific enolase promoter (Andersen, Eur J. CellBio 62 (1993), 324-332; Alouani, Hum. Gene Ther. 3 (1992), 487499); theMAP-1B promoter (Liu and Fischer, Gene 171 (1996), 307-308); the L1promoter (Chalepakis, DNA Cell Biol. 13 (1994), 891-900); the aromaticamino acid decarboxylase promoter (Le Van Thai, Mol. Brain Res. 17(1993), 227-238); the dopamine β-hydroxylase promoter (Mercer, Neuron 7(1991), 703-716); the NCAM promoter (Holst, J. Biol. Chem. 269 (1994),22245-22252); the HES-5 HLH protein promoter (Takebashi, J. Biol. Chem.270 (1995), 1342-1349); the α1-tubulin promoter (WO 95/25792, loc.cit.); the α-internexin promoter (Ching, J. Biol. Chem. 266 (1991),19459-19468); the peripherin promoter (Karpov, Biol. Cell 76 (1992),4348); the synapsin promoter (Chin, J. Biol. Chem. 269 (1994),18507-18513); the GAP-43 promoter (Starr, Brain Res. 638 (1994),211-220); the cyclic nucleotide phosphorylase I promoter (Scherer,Neuron 12 (1994), 1363-1375); the myelin basic protein promoter(Wrabetz, J. Neurosci. Res. 36 (1993), 455-471); the JC virus minimalcore promoter (Krebs, J. Virol. 69 (1995), 2434-2442); the proteolipidprotein promoter (Cambi and Kamholz, Neurochem. Res. 19 (1994),1055-1060); the cyclic nucleotide phosphorylase II promoter (Scherer,Neuron 12 (1994), 1363-1375). In order to provide means of identifyingoligodendrocytic precursor cells, WO 00/23571 proposes the use of apromoter which specifically drives expression in said oligodendrocytesor progenitor cells thereof. As specific promoters, the followingpromoters are disclosed: the CNP-P1 promoter, the CNP-P2 promoter, theCNP-P1+P2 promoter, the NCAM promoter, the myelin basic proteinpromoter, the JC virus minimal core promoter, the myelin-associatedglycoprotein promoter, the proteolipid protein promoter or the P/CNP2promoter. WO 01/53503 describes a method for enriching hippocampalneural progenitor cells by employing a promoter which specificallydrives expression in neural progenitor cell but not in other cells ofhippocampal tissue. Yet, WO 01/53503 proposes the use of Tα1 tubulinpromoter or nestin enhancer (Lothian, Eur. J. Neurosci. 9 (1997),452462). As pointed out herein above Tα1 tubulin promoter is also activein mature neurons and nestin is not only expressed in neuroepithelialcells but also in other cell populations (see Cai, Dev. Biol. 251(2002), 221-240). Furthermore, the intermediate filament nestin is alsoexpressed after traumatic injury of spinal cord or brain tissue; see,inter alia, Shibuya, Neurosci. 114 (2002), 905-916. In addition, nestinexpression persists in astrocytes (Schmid-Kastner, Int. J. Dev.Neurosci. 20 (2002), 29-38) and a large percentage of nestin-expressingcells have been proposed to be committed to astroglial cells (Wei, BrainRes. Dev. Brain Res. 139 (2002), 9-17). The neuron-specific enolasepromoter is often expressed in various tumors, and is found routinely inhistopathological analysis. Therefore the medical or diagnostic use ofthis promotor is risky since it may lead to an enrichment of cells whichare tumorigenic or an enrichment of poorly differentiated cells ofnon-neuronal origin; see Lee, Surg. Today 30(7) (2000), 658-662;Nakachi, J. Gastroenterol 35(8) 6 (2000), 31-634; Standop, Pancreas23(1) (2001), 36-39 or Muroi, Intern. Med. 39(10) (2000), 843-846.MAP-1B promoter is expressed at high level in mature neurons undergoingsynaptic rearrangement or sprouting. It is also highly expressed inmature neurons present in the dorsal root ganglia and the motor neuronsof the spinal cord, see Illing, Audiology and Neurootology 6 (2001),319-345 or Soares, Eur. J. Neurosci. 16(4) (2002), 593-606. The L1promotor leads to protein expression in mature neurons of the brain suchas Golgi, granule, basket and stellate cells of the cerebellum andpyramidal, granule, hilar interneurons in the hippocampus and ganglion,amacrine and horizontal cells in the retina; Rünker, J. Neurosci. 23(1)(2003), 277-286. In adult mammals, the aromatic amino acid decarboxylasepromoter is active in the dopaminergic and serotonergic neurons,therefore the promoter is merely active in mature, differentiatedneurons; Chatelin, Brain Res. Mol. Brain. Res. 97(2) (2001), 149-160.The dopamine beta-hydroxylase promoter is mainly active in dopaminergic,differentiated neurons, Matsushita, J. Neurochem. 82(2) (2002), 295-304.The NCAM promoter is not neuron-specific, since during in vitromyogenesis of muscle cells, an upregulation of some NCAM transcripts canbe observed. Furthermore, NCAM can also be expressed on Schwann cells;Roubin, Exp. Cell Res. 200(2) (1992), 500-505 or Dedkov, ActaNeuropathol 103(6) (2002), 565-574. HES-5 helix-loop-helix promoter is apromotor which is known to be active in cells differentiating intoastrocytes; Ohtsuka, J. Biol. Chem. 276(32) (2001), 30467-30474. In theadult CNS the alpha-internexin promoter is active mainly indifferentiated neurons of the brain, cerebellum and spinal cord. Inparticular, it is still active in the cerebellar granule cells; Ching,J. Neurosci. 19(8) (1999), 2974-2986 and Lavavasseur, Mol. Brain Res. 69(1999), 104-112. Peripherin promoter is still active in manydifferentiated, mature neurons of the peripheral nervous system. In thecentral nervous system, it is expressed in neurons with peripheralprojection. It can be reinduced in the central nervous system followingstab injury and cerebral ischemia; see, inter alia, Beaulieu. Brain Res.946 (2002), 153-161. Also the synapsin promoter is active in mature,differentiated neurons; see Chin, J. Biol. Chem. 269 (1994),18507-18513. Similarly, the GAP43 promoter is induced in regeneratingmature neurons; see Uvadia, Development 128(7) (2001), 1175-1182. Cyclicnucleotide phosphorylase I promoter, proteolipid protein promoter andmyelin basic protein promoter are activated and specific inoligodendrocytes; see Scherer, Neuron 12(6) (1994), 1365-1375; Wrabetz,J. Neurosci. Res. 36(4) (1993), 455471 and Cambi, Neurochem. Res. 19(8)(1994), 1055-1060. Similarly, cyclic nucleotide phosphorylase IIpromoter is active in oligodendrocyte precursors; Scherer, Neuron 12(6)(1994), 1365-1375. Also, the previously described JC virus minimal corepromoter is in vivo merely active in glial cells; see Krebs, J. Virol.69(4) (1995), 2432-2442.

In summary, to date there are neither a highly-specific markers nor generegulation sequences available which provide for either detection meansof neuronal restricted/determined cells or which would mediate specificexpression in such cells in mammalians, preferably in humans.

The discussion of the prior art herein above highlights the need formeans and methods for the detection and/or isolation of specific cellswhich are restricted to a neuronal fate, i.e. for the detection andisolation of neuronal restricted precursor cells. The difficultiesassociated with prior art methods discussed herein above andincompatibility with human tissue analysis confirm that novel, specificand quantifiable indicators of neurogenesis are desired. Considering thetherapeutic benefit of such cells and the constant need forcell-specific gene expression, the technical problem underlying thepresent invention is the provision as selective markers andlabeling-methods for neuronal determined cells in vivo and in vitro.

According to the invention, this problem is solved by the provision ofthe embodiments of the claims.

Thus, the present invention relates to the use of a regulatory sequencefor specific, early transient expression of a heterologous nucleotidesequence in proliferative neuronal determined cells, whereby saidregulatory sequence is selected from the group consisting of

-   -   (a) regulatory sequences comprising the nucleotide sequence        shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ        ID NO: 3 or as shown in SEQ ID NO: 4;    -   (b) regulatory sequences comprising the nucleotide sequence        contained in the insertion of clone DSM 15111 and obtainable by        amplification using two oligonucleotides having the sequences        indicated under SEQ ID NO: 9 and SEQ ID NO: 10;    -   (c) regulatory sequences comprising at least one nucleotide        sequence of SEQ ID NO: 1 from position 1166 to 1746, from        position 1166 to 2049, from position 1785 to 1843 or from        position 1953 to 2775;    -   (d) regulatory sequences comprising at least one nucleotide        sequence of SEQ ID NO: 2 from position 529 to 1079, from        position 529 to 1390, from position 1118 to 1175 or from        position 1291 to 2137;    -   (e) regulatory sequences comprising at least a functional part        of a sequence of (a) to (d) and causing specific expression in        neuronal determined cells;    -   (f) regulatory sequences comprising a nucleotide sequence which        is at least 75% identical to a sequence as defined in (a) to (d)        or which comprises a nucleotide sequence which is at least 78%        identical to the nucleotide sequence as shown in SEQ ID NO: 1        from position 1166 to 1746 or from position 1166 to 2049 or to        the nucleotide sequence shown in SEQ ID NO: 2 from position 529        to 1079, from position 529 to 1390 which comprises a nucleotide        sequence which is at least 82% identical to the nucleotide        sequence as shown in SEQ ID NO: 1 from position 1785 to 1843 or        to the nucleotide sequence as shown in SEQ ID NO: 2 from        position 1118 to 1175 or which comprises a nucleotide sequence        which is at least 75% identical to the nucleotide sequence as        shown in SEQ ID NO: 1 from position 1953 to 2775 or to the        nucleotide sequence as shown in SEQ ID NO: 2 from position 1291        to 2137; and    -   (g) regulating sequences comprising a nucleotide sequence which        hybridises with a complementary strand of the regulatory        sequence as defined in (a) to (f).

The regulatory sequences described herein are capable of impartingneuronal determined, cell-specific expression to nucleotide sequenceswhich are controlled by them. Furthermore, the herein describedregulatory sequence have the advantage that they are active inproliferating neuronal determined cells.

As documented in the appended examples the invention is based on thesurprising finding that doublecortin (DCX) is transiently and earlyexpressed in proliferating neuronal progenitor cells, newly generatedproliferating neuroblasts, migrating neuronal precursor cells as well asproliferating neuronal determined/restricted cells. All these cells leadto neurons, and the regulatory sequence as defined herein cansuccessfully drive expression of heterologous genes or coding sequencesin cells which are neuronal determined/neuronal restricted andmitotically active.

In this invention, it is shown that doublecortin acts as an indicatorfor adult neurogenesis, and the temporal expression pattern of DCX inneurogenic regions of the adult brain was determined. The invention asdocumented in the examples also relates to the fact that it can be shownthat when newly generated cells begin expressing mature neuronalmarkers, DCX immunoreactivity is sharply decreased below the level ofdetection and remains undetectable thereafter. This transient expressionpattern of DCX in proliferative neuronally committed progenitorcells/neuroblasts documents that DCX is a suitable marker for adultneurogenesis and provides, inter alia, for an alternative to BrdUlabeling. It is also observed and documented that the amount of cellsexpressing DCX is decreased with age, which coincides with the reductionof neurogenesis in the aging dentate gyrus. The results of the inventioncould only be obtained since the antibodies employed in this study arehighly specific for DCX. Furthermore, the examples convincingly showthat the regulatory sequence, relating to DCX expression in vivo and asdefined herein, is sufficient and specifically active in neuronaldetermined/neuronal restricted cells. As documented in the appendedexamples, in an adult transgenic mouse expressing EGFP under control ofthe regulatory sequence of the invention, high expression of EGFP isfound exclusively in neurogenic regions of the brain. Expression can notbe detected in organs other than the CNS, such as skin, muscle, gut,kidney, liver, heart, lung, etc. Within the neurogenic regions (dentategyrus of the hippocampus, ventricle wall, rostra-migratory stream andolfactory bulb), expression of EGFP largely overlaps with the endogenousexpression of DCX. Therefore, EGFP under control of the regulatorysequence as defined herein is expressed in neuronalrestricted/determined cells which are mitotically active. Furthermore,the morphology of EGFP expressing cells resembles that of young immatureneurons or of migrating neuronal precursors cells. In context of thisinvention, it can be shown that EGFP expression does not co-localizewith glial fibrillary acidic protein (GFAP), a marker for astrocytes,excluding a astroglial cell fate of EGFP positive cells. Similarly, theregulatory sequence of the invention does not drive expression of EGFPin HEK293 cells, a non-neuronal lineage cell type.

Neurogenesis is a process that involves the regulation of cellproliferation, determination and differentiation, in particular ofneuronal cells like, but not limited to,dopaminergic/cholinergic/GABA-ergic or noradrenergic neurons. Neuralstem cells are slowly dividing multipotent cells residing in neurogenicregions. They give rise to fast dividing neuronal determined precursorcells that by proliferation increase and potentiate the neurogenicactivity of a neural stem cell. After several rounds of division theyturn postmitotic and start to neuronally differentiate and to mature.Therefore, dynamic changes of the level of neurogenesis occur mainly atthe level of neuronal precursor proliferation. Cells that give rise tocells expressing DCX might have been proliferative or quiescent. Incontext of this invention, “quiescent” means currently not dividingcells which have the potential to divide in the future. The term“neurogenesis” as used herein describes all events that occur when cellsat one point of their life induce and start a neuronaldetermination/neuronal differentiation program that ultimately leads tothe acquisition of a neuronal phenotype. Said cells may be neural stemcells, but may also be other cells/cell types of the organism that havethe potential to acquire a neuronal phenotype. Corresponding examplesare given below and in the experimental part.

In accordance with this invention, the term “transient and earlyexpression”/“early transient expression” defines a period of timestarting after the moment that a cell has restricted to a neuronal fate,but before said cell expresses marker for mature neuronal cells/neurons,such as NeuN. In accordance with this intervention and documented in theexamples, the activity of the DCX regulatory sequence described hereinis progressively down-regulated concomitantly with neuronal maturation,defined by the appearance of mature neuronal markers, like NeuN.

As demonstrated in the experimental part, it could be shown that DCX isexpressed early, already during neuroblast proliferation, and longbefore (7 days) the onset of expression of NeuN in the rat dentategyrus. This is in sharp contrast to data from Kempermann, Development130 (2003), 391-399, which demonstrate that neurons acquire a maturephenotype (NeuN expression) right after cell birth (1 day after BrdUincorporation). Based on these data, Kempermann concludes that the useof DCX is not sufficient as a indicator of neurogenesis. Furthermore,and in contrast to this invention, Cooper-Kuhn (2002), loc. cit. showedthat DCX is not present in the proliferative pool of neuronalprecursors, but only in postmitotic, non-proliferative cells. However,for the uses, methods, genetically modified cells and non-humanorganisms provided in this invention it is particularly important thatDCX is expressed in proliferating cells. For example for the isolationof neuronally committed progenitor cells, for in vitro propagation, for(drug) screening procedures that search for compounds with the capacityto increase the pool of neuronally committent progenitor cells in vitroas well as for medical interventions in the adult brain as describedherein, it is important that proliferative, neuronally committed cellscapable of specifically expressing heterologous sequences (nucleic acidmolecules, genes, etc.) as described herein are employed. All these usesand methods, accordingly, require that the targeted cell type isproliferatively active.

The term “neuronal determined cell” relates to a cell/cell type thatwill (upon and during differentiation) exclusively lead, directly or viaits progeny, to neurons. It is also envisaged that said term relates tocells/cell types which, under experimental settings or under in vitroconditions selected, acquire a neuronal phenotype. An example of suchconditions is given in the appended experimental part relating toretinal pigmented epithelium (RPE) cells, wherein it is illustrated thatthe regulatory sequences described herein may be active in RPE cells andthat these cells have the capacity to acquire a neuronal phenotyp.Therefore, the term “neuronal determined cell” also comprises cellswhich are not necessarily of neuro-ectodermal origin, like inter alia,cells from the hematopoietic system, mesenchymal cells or ectodermalcells. Further definitions and explanations of the term “neuronaldetermined cell” are given herein below and in the experimental part.

The transient and early expression of the heterologous nucleotidesequence is specifically and/or uniquely observed in the above definedneuronal determined cells. Accordingly, the term “specific, early andtransient expression” means that the early and transient expression isdue to the activity of the regulatory sequence defined herein and thatsaid activity leads to the expression of a heterologous nucleic acidmolecule in neuronal determined cells.

The invention provides for a selection and marker system forneuronal-restricted cells, preferably neuronal-restricted precursorcells and for an indicator system for neurogenesis, preferably forneurogenesis occurring in a mammal postnatally, most preferably forneurogenesis in the mammalian adult brain. The findings as documented inthis invention are in clear contrast to results and interpretations inthe art. For example Kempermann (2003), loc. cit. is of the opinion thatit is not sufficient to use the neuronal marker doublecortin (DCX) asindicator of neurogenesis. In Kempermans work, the relative number ofnew neurons, as detected by BrdU/NeuN doublelabeling remains stableduring the period of investigation (1 day to 11 months after BrdUlabeling). Yet, Kempermann and co-workers miss the early time points(between 2 h and 7 days after single BrdU-injection) and, accordingly,the initial wave of BrdU/DCX positive cells that later turn intoBrdu/NeuN cells is not observed. Therefore the results presented hereinare in clear contrast to data from Kempermann, Development 130 (2003),391-399, which demonstrate that neurons acquire a mature phenotype (NeuNexpression) right after cell birth (1 day after BrdU incorporation).Based on these data, Kempermann concludes that the use of DCX is notsufficient as a indicator of neurogenesis.

Similarly, Nacher, European Journal of Neuroscience 14 (2001). 629-644,teaches that DCX is expressed and found in cells within areas, where “noadult neuronal migration occurs”. Nacher concludes that DCX is expressedin differentiated neurons and speculates “that DCX expression indifferentiated neurons could be related to its capacity for microtubulereorganization”. In contrast to Nacher (2001), the invention and theappended examples document that DCX-downregulation in newly bornneuronal determined cells coincides with the maturation into neurons andthat in matured neurons, DCX is not detected. Therefore, the prior artwas neither able to relate the DCX expressing cell population to thelater NeuN positive mature neurons nor to the fact that DCX is merelytransiently expressed early in neuronally committed proliferatingprogenitor cells. The prior art was not able to relate DCX expression tothe proliferating pool of neuronally determined precursor cells. At themost, the prior art, like Nacher (2001), loc.cit. and Cooper-Kuhn(2002), loc. cit., has speculated that DCX may be a marker for“immature”, “young” and/or “migrating” non-proliferative andpost-mitotic neurons or even non-neuronal cells, such as glial cells. Incontrast, the present invention shows that DCX-promotor drivenexpression is a useful transient marker for early neurogenic events suchas neuronal commitment and neurogenic proliferation and can besuccessfully be employed in the uses and methods provided herein.

The prior art considered DCX as a marker specific for postmitotic,non-proliferative young neurons, i.e. postmitotic cells which lostirreversibly their capacity to divide. Yet, and in contrast to the priorart it is documented herein and in particular in the appended examplesthat DCX is expressed already in the proliferating pool of neuronalprecursor cells. This surprising finding does not only allow thedetection of these neuronal restricted/determined cells and,accordingly, the detection of dynamic changes in neurogenesis but alsoprovides for unique tools for the detection/identification and/orverification of neurogenetic substance; as will be described hereinbelow.

Furthermore, the invention shows that neither DCX immunoreactivity norEGFP expression under control of the human DCX regulatory sequencedescribed herein does co-localize with glial fibrillary acidic protein(GFAP), a marker for astrocytes, excluding an astroglial cell fate ofcells expressing DCX protein or EGFP under the herein defined regulatorysequences. The invention documents in addition that DCX is not expressedin glial cells, such as astrocytes or oligodendrocytes, which is inclear contrast to the notion by (Nacher, Europ. J. Neurosc. 14 (2001),629-644) that DCX is also expressed in glial cells.

With the provision of the specific uses of the regulatory sequencesprovided herein, it is now possible to detect and/or selectneuronal-determined cells (i.e. newly generated neurons) in vivo and invitro. Accordingly, the present invention provides for specific uses(detailed herein below) for said regulatory sequences as well as foruseful host cells and host organisms, like transgenic non-human animals,which comprise said regulatory sequences and are particularly useful inmethods described herein below. In contrast to methods in the prior artemploying immunohistological approaches which comprise detrimentalfixation and/or permeabilization steps, the invention now provides formeans wherein neuronal progenitor/stem cell activity may be determined,measured and analyzed without fixation. Accordingly, the presentinvention provides for methods for in vitro and in vivo detecting andselecting neuronal-restricted precursor cells, deriving, inter alia,from organs and larger population of mixed cell types from mammals,preferably from mouse or rat, most preferably from human. Therefore, theinvention also comprises specific uses of a regulatory nucleic acidsequence defined herein. The inventive regulatory sequences may be used,inter alia, to isolate neuronal-restricted proliferative cells, to alterthe biology of neuronal-restricted cells, as well as to identifyneuronal-restricted cells within a large population of mixed cell types,both in vitro and in vivo. Preferably said cells are neuronalrestricted/determined progenitor cells. The specific neurogenesismarker/indicator provided herein and documented in the appended examplesare also useful for the analysis of potential therapeutics and thediscovery of drug targets to stimulate endogenous CNS stem cells inorder to generate new nerve cells in the CNS. In addition, such agenetic indicator provided herein allows the in vitro and in vivodetection and isolation of any cell in an organ or larger population ofmixed cell types that is in the process of acquiring a neuronalphenotype. Therefore, such a marker/indicator system provided hereinallows the detection means for processes associated with neuronaltransdifferentiation. The corresponding embodiments for these uses andmethods are provided herein below.

The term “regulatory sequence” refers to nucleotide sequences whichinfluence the expression level of a nucleic acid molecule, e.g. a gene(or of a nucleic acid sequence coding for an, inter alia, antisensemolecule), for instance by rendering expression tissue- or cellspecific. In this sense, regulatory sequences are understood to meanelements hereinafter also called regulatory elements, which impart to apromotor, preferably to a minimal promoter, additional expressionproperties. In the context of the invention, the term “promoter” refersto nucleotide sequences which are necessary to initiate transcription,that is to say to bind RNA polymerase, and for instance contain the TATAbox or a TATA-box like motif. Moreover, the term “regulatory sequence”may also comprise sequences outside the 5′-flanking promoter region.Such sequences are functional in both orientations and are less fixed intheir position than promoters, they are preferably within the region ofthe non-translated sequences of the mammalian DCX gene, preferably mouseor human, as they are described within the framework of the presentinvention (SEQ ID NOs. 1 to 4). Such sequence elements may includeenhancers, silencers as well as further modulators which may regulatethe expression of a gene up or down or which may render the promotordescribed herein inducible. Enhancers or silencers are often located inintrons or in the 3′-flanking region of a gene. The regulatory sequencemay also be a promoter which within the meaning of the invention ischaracterized by exerting all functions of a promoter, that is to sayinitiation of RNA polymerization, mediation of a specific expressionstrength and regulation of expression, preferably depending on the celltype, especially preferably with specificity for neuronaldetermined/neuronal restricted cells, preferably neuronaldetermined/restricted progenitor cells. The sequences represented in SEQID NOs. 1 to 4 or the above-defined segments of said sequences areregulatory sequences which at the same time correspond to the definitionof a promoter.

An example of such a regulatory sequence in accordance with thisinvention is the insert of deposited clone DSM 15111. DSM 15111 is theplasmid pEGFP-N1 (from Clontech, GenBank U55762; with deleted CMVpromotor) comprising an insert corresponding to nucleotide 1 tonucleotide 3509 of SEQ ID NO. 1, and referenced by the depositor asSTBL2-phuDCXpromoEGFP1. DSM 15111 was deposited at the Deutsche Sammlungvon Microorganismen und Zellkulturen GmbH, Braunschweig, Germany on Jul.25, 2002. Said deposit was made in by University of Regensburg(Klinikum), Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany. Theinsert of deposited clone comprises the human regulatory sequence of DCXas defined herein above may be obtained by methods known in the art,which comprise, e.g. PCR-reactions. For example, with the use ofoligonucleotides, for instance sequences as indicated under SEQ ID NOs.9 and 10, the complete promoter/regulatory sequence of the human DCXgene can be amplified; see also appended Example II. Said twooligonucleotides (oligo no:1: AAC ACC TAT TAA TGC CCA; SEQ ID NO.: 9 andoligo no:2: TCA GAG ACC TGA GCG TGG GAG AA; SEQ ID NO.: 10) may also beemployed to obtain a regulatory sequence of the present invention fromthe deposited clone DSM 15111. By means of oligonucleotide pairs5′-deleted or 3′-deleted fragments of the human DCX regulatory sequenceswhich still allow neuronal determined cell-specific expression, can beamplified and obtained. By provision of the sequences disclosed herein,the person skilled is readily in a position to deduce correspondingoligonucleotides. Accordingly, oligonucleotide pairs which enable askilled person to amplify the corresponding promoter fragments from thedeposited clone can be derived from the nucleotide sequences indicatedin SEQ ID NOs. 1 to 4. When the regulatory sequences of the inventionare provided from the human DCX regulatory sequence by amplificationfrom clone DSM15111, the specificity of the PCR reaction can beincreased by a preceding additional PCR reaction. Moreover, the sequenceof the promoter fragments can be detected by direct sequencing with thedeposited clone serving as a template. To this end, a skilled person canderive sequencing primers from the nucleotide sequences indicated underSEQ ID NOs. 1 to 4.

The invention is based on the finding that transient, early DCXexpression—provides for means for the detection and isolation ofneuronal determined and proliferative cells and that an about 3.5 kbpromoter fragment of the human DCX gene (FIG. 9 and SEQ ID NO. 1,respectively) cloned upstream of the coding region of a reporter gene(green fluorescent protein, EGFP, or DsRed2) led to the expression ofsaid reporter gene in, inter alia, neuronal precursor cells fromdissociated telencephalon (mouse) cultures, in young neuroblasts ofhuman fetal cortical stem cell cultures (see appended examples) or incultured cells, like day 10 to 14 mouse embryonic forehead cells. Inaddition, it could be documented that non-human transgenic animalsexpressing marker genes (e.g. EGFP) under the control of the regulatorysequences disclosed herein, only show marker-gene expression in newlygenerated, neuronal determined/restricted precursors, in particular inbrain regions involved in active neurogenesis. So far, the prior art hasnot provided sequences or means which bring about the above describedspecificity of DCX expression. The specificity forneuronal-restricted/neuronal determined cells of the regulatorysequences functional parts thereof can be proven, inter alia, bydeletion and transfections studies provided herein. For example,transfection of recombinant molecules, comprising a regulatory sequence(or a functional fragment or part thereof) of the present invention willnot lead to a expressed (marker/reporter) sequence in non-neuronalcells, like fibroblasts or HEK293 cells. Yet, a correspondingtransfection into cells which are neuronal precursor cells, liketransfection into cultured day 10 to day 14.5 embryonic mouse forebraincells, leads to an activation of the regulatory sequence as disclosedherein and the corresponding marker/reporter sequence is expressed.Therefore, the skilled artisan is in a position to deduce, inter alia,functional parts or fragments of the inventive regulatory sequences.

Isolation of partial sequences from one of the above-describedregulatory sequences can be achieved by standard molecular-biologicalmethods known to a skilled person, for instance according to Sambrook(Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. (1989)). This sourcecan also be drawn on for all other molecular-biological techniquesmentioned in the present description. In order to test the isolatedfragments for cell-specificity in neuronal restricted cells, the methodsdescribed in the examples and herein below can be used. For thispurpose, the fragments of sequences as defined above, are, inter alia,cloned into expression vectors comprising an additional markergene/reporter gene and, in parallel experiments, the expression of areporter gene in neuronal-restricted cells and in cells not expressingDCX or cells which are not or are not yet neuronal-restricted/determined(for instance, still pluripotent stem cells, glial precursor cells,glioblasts, astrocytes, mature neurons, non-nervous system cells, likefibroblasts or epithelial cells). Subsequently, it is measured in(transient) assays whether the fragment of the regulatory sequencedisclosed herein leads to a specific expression of the marker/reportergene in the neuronal restricted/determined cell. Specific expression inneuronal-restricted cells within the meaning of the invention is, e.g.,acknowledged if the level of expression compared to the cells notexpressing DCX or the corresponding marker gene is increased at least5-fold, preferably at least 8-fold, especially preferably at least10-fold, particularly preferably at least 15-fold and most preferably atleast 20-fold.

Functional fragments of the regulator sequences to be employed inaccordance with the present invention preferably comprise, but are notlimited to, regulatory sequences comprising at least one nucleotidesequence of SEQ ID NO: 1 from position 1166 to 1746 (or from position1166 to 2049), from position 1785 to 1843 or from position 1953 to 2775and/or at least one nucleotide sequence of SEQ ID NO: 2 from position529 to 1079 (or from position 529 to 1390), from position 1118 to 1175or from position 1291 to 2137. However, in a more preferred embodimentof the present invention, the functional fragment of the regulatorysequence for DCX expression (i.e, the regulatory sequence capable ofcausing early transient expression of heterologous nucleotide sequencesin neuronal determined cells) comprises or is the nucleotide sequence asshown SEQ ID. NO. 1 from nucleotides 1166 to 1746 or from nucleotides1166 to 2049 and from nucleotides 1953-2775 (corresponding to “regions 2and 4” as defined in appended FIG. 12). Similarly, said regulatorysequence may comprise or may be the nucleotides as shown SEQ ID. NO. 2from nucleotides 529-1079 or from nucleotides 529 to 1390 and fromnucleotides 1291-2137 (also corresponding to “region 2 and 4” as definedin appended FIG. 12). In a most preferred embodiment of the presentinvention, the regulatory sequence to be employed is the nucleotidesequence defined as “region 2” herein and in the appended examples,whereby said region may also (and in addition) comprise the following“exon” or “exons”. Accordingly, the regulatory sequence to be employed,in one embodiment, is or comprises nucleotides 1166 to 1746 (or 1166 to2049) of SEQ ID NO. 1 or is or comprises nucleotides 529 to 1079 (or 529to 1390). In this context, also nucleotide sequences which are at least78%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95% and mostpreferably at least 99% identical to the nucleotide sequence as shown inSEQ ID NO: 1 from position 1166 to 1746 (or 1166 to 2049) or to thenucleotide sequence shown in SEQ ID NO: 2 from position 529 to 1079 (or529 to 1390) and/or nucleotide sequences which are at least 75% morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95% and most preferably at least99% identical to the nucleotide sequence as shown in SEQ ID NO: 1 fromposition 1953 to 2775 or to the nucleotide sequence as shown in SEQ IDNO: 2 from position 1291 to 2137 may be comprised in a regulatorysequence that causes/drives expression in neuronal determined cells. Itis also envisaged that the regulatory sequence merely comprises thenucleotide sequences as shown in the herein defined regions 2 and 4 (seeFIG. 12) or homologues thereof which are at least 78% more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 95% and most preferably at least 99% identicalto the regions 2 and 4 as defined herein. Said regions 2 and 4 may bedirectly linked on the nucleotide level but it is also envisaged thatsaid regions are separated by a spacer sequence, which may be the spaceras shown between these two regions in SEQ ID Nos. 1 and 2 (or asdepicted as “region 3 and exon 1 and 2) in FIG. 12 or which may be, morepreferably unrelated to the spacer as shown in SEQ ID NOs 1 or 2 (and inFIG. 12). The prior art has provided and deposited a nucleotide sequence“AL450490” in Genbank. Yet, in contrast to the present invention, inAL450490 a gene defined as “DCX gene” comprises merely the coding regionof DCX and a short 5′ segment of 748 nucleotides upstream of the ATGpresent in exon 4.

Another aspect of the invention relates to the above defined uses ofregulatory sequences which hybridize to one of the above-describedregulatory sequences of the invention, preferably to the complementarystrand thereof, and cause neuronal restricted/determined cell-specificexpression of a nucleotide sequence controlled by them.

These hybridizing sequences may be promoters as defined above orregulatory elements imparting neuronal restricted/determinedcell-specificity to minimal promoters.

The term “hybridize” as used refers to conventional hybridizationconditions, preferably to hybridization conditions at which 5×SSPE, 1%SDS, 1×Denhardts solution is used as a solution and/or hybridizationtemperatures are between 35° C. and 70° C., preferably 65° C. Afterhybridization, washing is preferably carried out first with 2×SSC, 1%SDS and subsequently with 0.2×SSC at temperatures between 35° C. and 70°C., preferably at 65° C. (regarding the definition of SSPE, SSC andDenhardts solution see Sambrook, loc. cit.). Stringent hybridizationconditions as for instance described in Sambrook, supra, areparticularly preferred. Particularly preferred stringent hybridizationconditions are for instance present if hybridization and washing occurat 65° C. as indicated above. Non-stringent hybridization conditions,for instance with hybridization and washing carried out at 45° C. areless preferred and at 35° C. even less.

Such regulatory sequences preferably show a homology, determined bysequence identity, of least 75%, preferably of at least 80%, morepreferably of at least 85%, even more preferably of at least 90% andmost preferably of at least 95% to a sequence as defined in (a) to (d)herein above. Yet, the invention also relates to regulatory sequenceswhich comprise or is a nucleotide sequence which is at least 78%, morepreferably at least 80%, even more preferably at least 90% and mostpreferably at least 95% identical to the nucleotide sequence as shown inSEQ ID NO: 1 from position 1166 to 1746 (or from position 1166 to 2049)or to the nucleotide sequence shown in SEQ ID NO: 2 from position 529 to1079 (or from position 529 to 1390). Also envisaged are regulatorysequences which comprises or is a nucleotide sequence which is at least82%, more preferably at least 85%, even more preferably at least 90% andmost preferably at least 95% identical to the nucleotide sequence asshown in SEQ ID NO: 1 from position 1785 to 1843 or to the nucleotidesequence as shown in SEQ ID NO: 2 from position 1118 to 1175. Similarly,the invention provides for uses of regulatory sequences which comprise anucleotide sequence which is at least 75%, more preferably at least 80%,even more preferably at least 90% and most preferably at least 95%identical to the nucleotide sequence as shown in SEQ ID NO: 1 fromposition 1953 to 2775 or to the nucleotide sequence as shown in SEQ IDNO: 2 from position 1291 to 2137.

The regulatory sequences to be employed in accordance with thisinvention preferably show a homology, determined by sequence identity,of at least 50%, preferably at least 60%, particularly preferably atleast 70%, advantageously at least 80%, preferably at least 90% andespecially preferably at least 95% to the sequences indicated under SEQID NO 1 to 4, preferably over the entire length of the sequencescompared. The hybridizing sequences are preferably fragments having alength of at least 100, more preferably at least 200, more preferably atleast 300, more preferably at lest 400 and most preferably at least 500nucleotides which have an identity of at least 75%, preferably at least80%, especially preferably at least 90% and particularly preferably atleast 95% with the sequence shown under SEQ ID NO. 1, 2, 3 or 4,respectively. If two sequences which are to be compared with each otherdiffer in length, sequence identity preferably relates to the percentageof the nucleotide residues of the shorter sequence which are identicalwith the nucleotide residues of the longer sequence. Sequence identitycan be determined conventionally with the use of computer programs suchas the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive Madison, Wis. 53711). Bestfit utilizes the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2(1981), 482489, in order to find the segment having the highest sequenceidentity between two sequences. When using Bestfit or another sequencealignment program to determine whether a particular sequence has forinstance 95% identity with a reference sequence of the presentinvention, the parameters are preferably so adjusted that the percentageof identity is calculated over the entire length of the referencesequence and that homology gaps of up to 5% of the total number of thenucleotides in the reference sequence are permitted. When using Bestfit,the so-called optional parameters are preferably left at their preset(“default”) values. The deviations appearing in the comparison between agiven sequence and the above-described sequences of the invention may becaused for instance by addition, deletion, substitution, insertion orrecombination. Such a sequence comparison can preferably also be carriedout with the program “fasta20u66” (version 2.0u66, September 1998 byWilliam R. Pearson and the University of Virginia; see also W. R.Pearson, Methods in Enzymology 183 (1990), 63-98, appended examples andhttp://workbench.sdsc.edu/). For this purpose, the “default” parametersettings may be used.

The techniques described in the appended examples may, inter alia, beused to determine whether hybridizing sequences mediate neuronaldetermined/neuronal-restricted cell-specific expression.

As pointed out above, the term “neuronal determined cell” relates, inaccordance with this invention to cells which differentiate into neuronsor which divide into neuronal restricted cells, i.e. into cells thatgenerate merely neurons. Accordingly, the term “neuronal determinedcell” as employed herein also comprises “neuronal restricted cells”.Therefore, the “neuronal determined/restricted cells” as described inthis invention lead during their differentiation or development merelyand exclusively to neurons and not to other cells of the nervous system,like, oligodendrocytes, astrocytes, Schwann cells, microglia cells, gliacells or even cells like endothial cells, fibroblasts and the like.However, the term “neuronal determined/restricted cell” does alsocomprise progenitor or stem cells, which activate the regulatorysequence of the present invention and, thereby, are on arestricted/determined differentiation/developmental pathway leading to(a) neuron(s). Stem cells can proliferate to give rise to daughter cellsidentical to the mother cells and thereby enlarge the stem cellpopulation. Alternatively, they can divide into various cell types. Astem cell that can produce any cell type of an organism, for example anembryonic stem cell, is referred to as totipotent, whereas a stem cellthat can produce only a defined subset of cell types is referred to aspluripotent. In several organs and systems, somatic stem cells have beendescribed. These somatic stem cells have the potential to divide intodaughter cells identical to the mother cells and thereby renew orenlarge the somatic stem cell population. Alternatively, a daughter cellcan differentiate into various cell types relevant to this organ/system.For example, neural stem cells have been described in the centralnervous system (CNS). These cells can differentiate into neurons,astrocytes and oligodendrocytes, i.e., the three major CNS cell types.The capacity of more or less differentiated cells to generate cell typespresent in another organ/system is referred to as trans-differentiation.Accordingly, the term “neuronal restricted/determined cell” as employedherein also refers to cells, like stem cells or trans-differentiatingcells, which comprise an activated regulatory sequence of the presentinvention. Yet, the term “neuronal determined/restricted cell(s)” asused herein is to be delimited from multi/pluripotent undifferentiatedstem cells that have not and or will not activate the regulatorysequence described herein. The term “neuronal restricted/determinedcell” refers, therefore, to any cell/cell type that will exclusivelylead, directly or via its progeny, to neurons in vivo or underexperimental or in vitro settings/conditions selected. The term“neuronal restricted/determined cell” also comprises migrating cells,like migrating neuronal precursor cells. By providing the regulatorysequences of doublecortin (DCX), the present invention provides for adistinct tool to detect, track, select and/or isolate “neuronalrestricted/determined cells”. Accordingly, the present inventionprovides for specific advantageous uses and methods, wherein theregulatory sequences described herein or host cells/host organisms (liketransgenic non-human animals, as well as organs, tissues or cells ofsuch organisms) comprising the regulatory sequences or recombinantnucleic acid molecules as defined herein, are employed.

In a particular preferred embodiment, the regulatory sequence of thepresent invention is of human, rat or mouse origin. Corresponding humanand mouse sequences are illustrated in SEQ ID NO.1 and 2. The regulatorysequences of the invention are preferably DNA or RNA molecules, the DNAmolecules being preferably genomic DNA.

The invention also relates to uses of recombinant nucleic acid moleculescomprising the regulatory sequence described herein. Said recombinantnucleic acid molecule comprises the regulatory sequences in an“isolated” form, preferably in combination with a heterologous nucleicacid sequence to be expressed. As used herein, the term “isolated” whenused in conjunction with a nucleic acid molecule/regulatory sequences ofthe present invention refers to a nucleic acid molecule which has beenseparated from an organism in a substantially purified form (i.e.substantially free of other substances originating from that organism),or a nucleic acid molecule having the same nucleotide sequence but notnecessarily separated from the organism (i.e. synthesized orrecombinantly produced nucleic acid molecules).

Preferably and most envisaged, the regulatory sequences described in thepresent invention are operatively linked to additional, heterologousnucleic acid sequences in a recombinant nucleic acid molecule. Asdetailed herein below, said additional nucleic acid sequence may be acoding gene as well as a nucleic acid sequence which, upon expression,leads to the production of an antisense construct, a ribozyme or thelike.

As employed herein, the term “heterologous nucleic acid molecule”, meansa nucleic acid molecule which is preferably operatively linked to theregulatory sequence described above but is not a nucleic acid moleculewhich codes for doublecortin (DCX) or a fragment thereof. Therefore,said “heterologous nucleic acid molecule” originates from a differentgenetic context than the regulatory sequence described above.Non-limiting examples of such “heterologous nucleic acid molecules” aregiven herein below and comprise in particular marker molecules, likeluciferase, galactosidase, GFP, EGFP, DsRed, etc. or tag-molecules, likeFlag-tags, CBP and others. Yet, as detailed below, also receptor genes,anti-apoptotic genes, genes coding for determination/differentiationmolecules, trophic factors, surface proteins, transcription factors,molecules directing neuronal migration or guidance are envisaged. Alsonucleic acid molecules which do not encode proteins are envisaged as“heterologous nucleic acid molecules”. Such nucleic acids comprise, butare not limited to, anti-sense molecules, aptamers, ribozymes,inhibiting RNA molecules and the like.

The term “recombinant nucleic acid molecule” relates to nucleic acidmolecules originating from a different genetic context and combined bymolecular biological methods. Here, the term “different genetic context”relates to genomes from different species, varieties or individuals ordifferent positions within a genome. Recombinant nucleic acid moleculescan contain not only natural sequences but also sequences, which,compared to the natural ones are mutated or chemically modified or else,the sequences are altogether newly synthesized sequences.

The recombinant nucleic acid molecules of the invention show one or moreof the above-described regulatory sequences in combination withsequences from another genetic context. An example of a recombinantnucleic acid molecule contains one or more regulatory sequences of theinvention or a minimal promoter derived and obtainable from thesequences disclosed herein in combination with a gene other than the DCXgene, preferably other than the human doublecortin gene. The term“recombinant nucleic acid molecule”, therefore, does not relate to anucleic acid molecules which comprises a DCX coding sequence under thecontrol of the regulatory sequences provided herein. The regulatorysequences are regulatory promoter elements that impart a neuronaldetermined/neuronal-restricted cell-specific expression.

Moreover, the recombinant nucleic acid molecules can contain, apart froma promoter containing one or more regulatory sequences of the invention,a polylinker sequence located downstream thereof and comprising one ormore restriction sites into which nucleotide sequences can be cloned bymethods known to a skilled person, which thus come under the expressioncontrol of the promoter. Said polylinker lies preferably in a regionthat is situated directly behind the transcription starting pointdefined by the promoter.

Furthermore, the recombinant nucleic acid molecule described herein maycontain a transcription termination signal downstream of the polylinker.Examples of suitable termination signals are described in the state ofthe art. The termination signal can, for instance, be the thymidinekinase polyadenylation signal. The herein-described recombinant nucleicacid molecules which preferably contain a nucleotide sequence to beexpressed can be directly employed for uses within the meaning of theinvention, such as DNA transfections, the generation of geneticallymodified host cells or non-human transgenic animals. Furthermore, saidrecombinant molecules can be employed in screening methods describedherein as well as in medical and scientific settings. The recombinantnucleic acid molecules of the invention may, also, be multiplied byconventional in-vitro amplifications techniques, for instance PCR.However, they can also conventionally be multiplied in vivo in a vector,and after nucleic acid preparation and subsequent removal from thevector, for instance by restriction cleavage, can be provided for usesrequiring for instance linearized expression units. Recombinant nucleicacid molecules, which preferably contain a nucleotide sequence to beexpressed, can also constitute expression units which are oftendesignated expression cassettes which can be easily cloned intodifferent standard vectors and depending on the vector can thus exertdifferent functions.

In a particular preferred embodiment, the recombinant nucleic acidmolecule of the invention comprises the regulatory sequence of theinvention which controls the expression of the additional nucleotidesequence.

Preferably, in context of this invention, the additional nucleotidesequence to be expressed is engineered “near” or in a certain distanceto the 3′-end of the regulatory sequences defined herein. “Near” meansthat the additional nucleotide sequence(s) or parts thereof is(are)cloned directly or at a certain distance to the afore-mentionedregulatory sequences, upstream, downstream or intermittently. Cloning iscarried out, however, preferably downstream because, as is known, theafore-mentioned regulatory sequences (promoters or functional partsthereof, require relatively defined distances from the transcriptionstarting point and from the TATA box, respectively, for their way offunctioning, that is to say for binding RNA polymerase or transcriptionfactors. “At a certain distance” means a distance which is suitable toallow silencers or enhancers to exert their function. Correspondingexamples are documented herein below, for example the engineering ofconstructs expressing selectable marker genes. It is of note that it isalso envisaged that partial sequences of the above defined regulatorysequences be employed in context of this invention, i.e. thatrecombinant nucleic acid molecules are constructed which comprise onepartial sequences or functional fragments of the regulatory sequencesfor DCX as defined herein. Such constructs may also be tested in theassays provided herein, e.g. transient expression assays for theirexpression function and level. For example, a construct comprising apartial sequence of the inventive regulatory sequence, and forcomparison purposes, the same construct without said partial sequence,can be analyzed in a transient expression assay in cultured cells,(preferably a primary cell, like embryonic mouse day 12 neuronalprecursor cells derived from forehead) and in cells not capable of ornot expressing DCX (like HEK293 cells). If the difference of theexpression level between cells capable of not expressing DCX is greaterin the case of the construct with the partial sequence than in the caseof the construct without the partial piece, then this partial sequencecomprises a functional silencer or enhancer element. Additionaltechniques for delimiting such elements are available to a skilledperson. A detailed example for such a technique is as follows: it is,inter alia, possible to deduce the minimal promoter element in theregulatory sequence described herein, by the construction of deletionmutants that express one of the fluorescent or luminescent proteins e.g.EGFP or DSRed etc. The minimal promoter should be sufficient andnecessary to drive expression of a gene in neuronal precursor cells,neuronal determined/restricted cells. Preferred minimal promoterscomprise either alone or in combination the “region 2” and/or the“region 4” as defined in FIG. 12, corresponding to the nucleotidesequence of SEQ ID NO: 1 from position 1166 to 1746 (or from position1166 to 2049) and from position 1953 to 2775 or the nucleotide sequenceof SEQ ID NO: 2 from position 529 to 1079 (or from position 529 to 1390)and from position 1291 to 2137. Said desired minimal regulatory sequencedoes, preferably and in one embodiment, not comprise the nucleotidesequence comprising position 3169 to 3501/3509 of SEQ ID NO. 1, does notcomprise the nucleotide sequence comprising position 2525 to 2859 of SEQID NO. 2 or does not comprise homologues thereof. These nucleotidesequences correspond to “region 5” as defined in FIG. 12. Yet, it is ofnote that regulatory sequence to be employed in the uses and methods ofthe present invention, as characterized herein, may also comprise said“region 5”. A further optional part of the regulatory sequence describedherein and to be employed is the “region 1” as defined in appended FIG.12.

Functional constructs of the regulatory sequence causing transient,specific expression in neuronal determined cell may be, e.g., tested bytransfection, into neuronal precursor cells derived from mouse embryonicday 10 to 14 forebrain and analyzed for expression as demonstrated inthe appended examples. Transfectons in non-neuronal cells e.g. HEK293cells serve as negative controls, since the functional promoter shouldnot be active in non-neuronal cells. In addition, transfections may bedone using EGFP positive cells derived from the transgenic animal(Example 4). A functional promoter/promoter fragment should be activedin these cells, however should be inactive in EGFP-negative cells fromthe same animals. Corresponding working examples are appended.

The embodiment comprises the above-described regulatory sequences whichare combined with at least one nucleotide sequence, which can beprovided by amplification from the insertion of the deposited cloneDSM15111, using for instance PCR, or parts thereof. Such an additionalsequence in the deposited clone DSM1511 encodes an enhanced greenfluorescent protein. For amplification of said additionalgene/nucleotide sequence (here EGFP), for instance pairs ofoligonucleotides can be used. The sequences of indicated by the SEQ IDNOs: 13 and 14, i.e. the sequences ATG GTG AGC MG GGC GAG GAG (SEQ IDNo:13) or CTT GTA CAG CTC GTC CAT GCC (SEQ ID No:14) may be employed toamplify the EGFP in said DSM 15111 clone. Accordingly, the additionalsequence coding for EGFP in deposited clone DSM 15111 may be replaced byany other desired sequence.

In accordance with this invention, it is also envisaged that more thanone additional sequence is comprised in the recombinant nucleic acidmolecule described herein. Accordingly, the invention also relates tothe use of recombinant nucleic acid molecules which comprise, besidesthe DCX regulatory sequences, clusters genes, preferably in the samereading frame. For example, two nucleotide sequences can be locatedbehind one another in one reading frame, that is to say, beingtranslationally fused (if both nucleotide sequences encode a protein ora fragment thereof). These coding regions can be directly adjacent toone another or can be spaced apart by a spacer. A spacer separates thetertiary structure of the two proteins spatially from one another, inorder to prevent their tertiary structures from negatively interacting.The spacer has, however, preferably the function of acting as a point ofattack for a protease, preferably an endogenous protease of thetransfected cell, with the result that the expressed proteins areseparated in vivo. Alternatively, the spacer can contain an IRESsequence (internal ribosomal entry site). This allows both genes to betranscribed under the control of a single promoter, their translationoccurring separately.

On the other hand, the two nucleotide sequences can also be encodedtranscriptionally independently from each other. For this purpose, eachnucleotide sequence is under the control of its own promoter, with atleast one promoter, preferably both promoters, comprising the regulatorysequences of the present invention.

Such an embodiment would allow the particular advantages ofco-transfection with expression constructs encoding an differentproteins or fragments thereof.

Another preferred embodiment of the invention relates to uses of theabove-described recombinant nucleic acid molecules or vectors, wherebythese recombinant nucleic acid molecules additionally contain/comprise anucleotide sequence to be expressed, wherein expression of thenucleotide sequence is controlled by the DCX regulatory sequence or aDCX promoter/enhancer comprising the regulatory sequence. In context ofthis invention, it is also envisaged that the regulatory sequence isemployed in its inducible form.

The “nucleotide sequences to be expressed” encode either a protein or(poly)peptide or RNA molecules which display their function on the RNAlevel. Nucleotide sequences encoding a protein, polypeptide or peptidecomprise a coding region which is characterized by a start codon (ATG),a sequence of base triplets encoding amino acids and a stop codon (TGA,TAG or TAA) if it concerns DNA. In the case of RNAs, the thymidine (T)is replaced with uracil (U). In the case of degenerated amino acidcodons, the base triplets can be adapted in accordance with the codonusage of the target cells, using prior art techniques. Examples ofnucleotide sequences which express RNA molecules are antisense RNA,inhibiting RNA, IRNA or ribozymes.

In a further embodiment, the invention relates to (a) recombinantnucleic acid molecule(s) described herein, wherein said nucleotidesequence to be expressed under the control of the regulatory sequence ofthe invention is a gene selected from the group consisting of a markeror receptor gene, an anti-apoptotic gene, adetermination/differentiation gene, a gene capable of inducing and/ordirecting neuronal migration or guidance, a gene encoding a tag, a geneencoding for a trophic factor, a gene encoding a surface protein, a geneencoding for a transcription factor, a gene encoding an enzyme orwherein said nucleotide sequence to be expressed is an antisensesequence or encodes for a ribozyme or an inhibiting/interfering RNAmolecule or the like. Accordingly, the invention relates in oneembodiment said marker or receptor gene to be expressed under thecontrol of the herein disclosed regulatory sequences are, e.g.,fluorescent proteins (e.g. green fluorescent protein) or proteins whichmay, directly or indirectly, lead to a visible or measurable signal,when expressed (e.g chloramphenicol acetyltransferase,beta-galactosidase).

Examples of marker or reporter genes, which allow the expressionactivity of regulatory sequences, preferably promoters, to be detected,preferably in eukaryotic cells, are described in the literature.Examples of reporter genes encode luciferase, (green/red) fluorescentprotein and variants thereof, like EGFP (enhanced green fluorescentprotein), RFP (red fluorescent protein, like DsRed or DsRed2), CFP (cyanfluorescent protein), BFP (blue green fluorescent protein), YFP (yellowfluorescent protein), β-galactosidase or chloramphenicolacetyltransferase, and the like.

For example, GFP can be from Aequorea victoria (U.S. Pat. No.5,491,084). A plasmid encoding the GFP of Aequorea victoria is availablefrom the ATCC Accession No. 87451. Other mutated forms of this GFPincluding, but not limited to, pRSGFP, EGFP, RFP/DsRed, and EYFP, BFP,YFP, among others, are commercially available from, inter alia, ClontechLaboratories, Inc. (Palo Alto, Calif.). For example, DsRed2 is alsoavailable from Clontech Laboratories, Inc. (Palo Alto, Calif.); seeappended examples. Also further luminescent proteins may be expressedunder the control of the regulatory sequence provided herein. In thiscontext, a nucleotide sequence coding, inter alia, for a protein of theluciferase family is envisaged.

The invention also relates to a recombinant nucleic acid moleculecomprising the regulatory sequence of the invention and a gene under itscontrol, whereby said gene encodes a tag. Said tag may be selected fromthe group consisting of a His-Tag, glutathione, a Strep-tag, a Flag-tag,CBP (Calmodulin-binding peptide), TAG-100 (available from Quiagen),E2-tag (from bovine papillomavirus type I transactivator protein E2) andZ-tag, but is not limited thereto. For example, the self-cleavablechitin-binding tag (e.g. from IMPACT-CN System) or influenzahemagglutinin (HA) may be employed in accordance with this invention. Itis also envisaged that the recombinant nucleic acid molecule of thisinvention is capable of expressing a peptide or protein sequence whichmay be detected by a specific antibody (or antibody fragment orderivative) directed against said peptide or protein.

It is furthermore one embodiment of the invention that the recombinantnucleic acid molecule to be used in accordance with the invention codesfor a (cell) surface protein, which may be detected by specificantibodies directed against said cell surface protein. A gene encoding asurface may for example be CD 24. This cell surface molecule wassuccessfully employed for the selection of transduced cells, seePawliuk, Blood 84 (1994), 2868-2877.

A gene encoding for a trophic factor and expressed under the control ofthe regulatory sequence of the present invention may be selected fromthe group consisting of NGF, BDNF, PDGF, NT-3, NT-4, NT-5, VEGF, PEDF,EGF, FGF, IGF, cardiotrophin, erythropoietin, leptin, LIF and TGF. Theserecombinant nucleic acid molecules are very useful in medical as well asscientific settings. For example, a NGF may be used in the treatment ofAlzheimer's Disease (Winkler, J. Mol. Med. 76(8) (1998), 555-567), NT-3in the treatment of Huntington's Disease (Perez-Navarro, J. Neurochem.75(5) (2000), 2190-2199) or Parkinson's Disease (Espejo, CellTransplant. 9(1) (2000), 45-53). Similarly, the expression of NT-4/5 mayhave beneficial effects in the treatment of Huntington's Disease(Perez-Navarro, J. Neurochem. 75(5) (2000), 2190-2199). VEGF may bedesired in the treatment of stroke (Jin, Proc. Natl. Acad. Sci. USA97(18) (2000), 10242-10247), whereas PEDF may have beneficial effects inMotor Neuron Diseases, like ALS (Bilak, J. Neuropathol. Exp. Neurol.58(7) (1999), 719-728). It is also described that EGF, FGF and PDGF havebeneficial effects in the treatment or prevention of stroke (Nakatomi,Cell. 110(4) (2002), 429-441 and Krupinski, Stroke 28(3) (1997),564-573). IGF was studied in stroke intervention (Liu., Neurosci, Lett.308(2) (2001), 91-94) and in the treatment of Huntington's Disease(Humbert, Dev Cell. 2(6) (2002), 831-837). Similarly, BDNF was employedin the medical intervention of Huntington's Disease (Perez-Navarro, J.Neurochem. 75(5) (2000), 2190-2199) and spinal cord injury (Hammond,Neuroreport 10(12) (1999), 2671-2675). GDNF was employed in Parkinson'sDisease (Nakajima, Brain Res. 916(1-2) (2001), 76-84; Espejo, CellTransplant. 9(1) (2000), 45-53) and HGF, like CNTF, were used in thetreatment of amyotrophic lateral sclerosis, ALS (Sun, J. Neurosci.22(15) (2002), 6537-6548 and Sendtner, Nature 358(6386) (1992),502-504). Cardiotrophin (Toth, J. Neuroscience Res. 69(5) (2002),622-632) protects PC12 cells against the excitatory damage, oxidativestress and apoptosis and erythropoietin (Juul, Acta. Peadiatr. Suppl.91(438) (2002), 36-42) was described to have neurotrophic andneuroprotective functions in the developing and injured brain. Leptin(Dicou, Neuroreport 12(18) (2001), 3947-3951) exerts neuroprotectionagainst toxicity, whereas LIF (Marzella, Hear. Res. 138(1-2) (1999),73-80) promotes survival of dissociated cultures of spinal ganglioncells. However, the use of the recombinant nucleic acid moleculeexpressing the trophic factors, especially the trophic factors listedabove is not limited by the distinct disorders mentioned above andfurther medical uses are envisaged.

In a further embodiment of the recombinant nucleic acid molecule of thepresent invention, the anti-apoptotic gene to be expressed under thecontrol of the inventive regulatory sequence is selected from the groupconsisting of bcl-2, Bm-3a, PTEN and (an) anti-caspase(s).Anti-apoptotic molecules and/or nucleic acid molecules inhibiting theapoptotic molecules, like caspases, such as antisense-caspase-3 areconsidered to reduce the cell death of neuronal determined cells. Thisis especially important, since apoptotic and neurogenic regions in theadult brain overlap (Biebl, Neurosci. Left. 291(1) (2000), 17-20).Promoting the survival of neuronal determined cells might raise thetotal population of neurons in the adult brain. Bm-3a, for example,activates the expression of Bcl-x(L) and Bcl-2 and promotes neuronalsurvival in vivo as well as in vitro (Smith, Mol. Cell. Neurosci. 17(3)(2001), 460470).

It is also envisaged that the determination/differentiation gene to beexpressed under the control of the regulatory sequence described hereinis the dopaminergic determination factor Nurr1. Nurr1 induces andpromotes dopaminergic differentiation of stem cells (Chung, Eur. J.Neurosci. 16(10) (2002), 1829-1838).

The gene capable of inducing and/or directing neuronal migration orguidance and being expressed under the control of the regulatorysequence of the present invention may, inter alia, be netrin;neuropilin, CXCR4, SDF-1, DCC, slit, robo, a semaphorin, a plexin familymember, an ephrin family member. These migration and/or guidancemolecules are well known in the art and relate, e.g. to moleculesattracting/repulsing growing axons in order to reestablish synapticconnection. Examples are CXCR4 and its ligand SDF-1 (Lu, PNAS 99(10)(2002), 7090-709. Without functional CXCR4, morphogenesis of thehippocampal DG (dentate gyrus) fails. In accordance with this invention,it is envisaged that loss of SDF-1/CXCR4 signaling could at least inpart, mediate this migratory defect. Semaphorin/plexin family membershave been described in Chen, Neuron 32 (2001), 249-263. For example, invivo, PlexA3 is crucial for proper targeting of a subset of hippocampalafferents, but less important for guidance of peripheral axon of thesuperior cervival ganglion and DRG. The slit/robo guidance system isdescribed in Zou, Cell 102 (2000), 363-375. Neuropilin receptors areexpressed by commissural neurons and are required to navigatecommissural axons across the midline of the CNS to their rostral targetsafter midline crossing. In this system, class 3 semaphorins act inconcert with another class of repellent proteins, the Slits; to preventcommissural axons from recrossing or lingering at the midline. A smallfamily of secreted proteins, termed netrins, which attract commissuralaxons before the midline crossing. Netrin-induced attraction is mediatedby the DCC (deleted colorectal cancer) family of receptors that includeFrazzled in Drosophila, UNC40 in C elegans and DCC and neurogenin invertebrates, see Yu, Nat Neurosci 4 (2001), 1169-1176. Ephins and ephrinreceptors (Eph) are also known in the art, see, e.g. Kullander, GenesDev 15 (2001), 877-888. The knockout of ephrinsB3, an EphA4 ligand,provided strong evidence that EphA4 interacts with ephrinsB3, expressedat the midline, to prevent CST axons from aberrantly recrossing themidline.

In a further embodiment of the invention, the gene encoding atranscription factor and being controlled by the regulatory sequencedescribed herein may be selected from the group consisting of NeuroD,BMP4, Nurr1 or ShcC.

The invention also provides for the above described uses of arecombinant nucleic acid molecule wherein an enzyme is expressed underthe control of the DCX regulatory sequence. A preferred example, besidesthe above mentioned reported genes like β-galactosidase or luciferase isCRE (CRE-recombinase). It is, inter alia, envisaged that CRE isexpressed under the control of the DCX regulatory sequence describedherein and such a construct is employed in the generation of a non-humantransgenic animal. Accordingly, the recombinant nucleic acid moleculemay be used for tranfection of, e.g. a zygote or an ES-cell, for thepreparation of said transgenic animal for use in recombination studiesand gene deletion systems. Such a CRE-positive transgenic animals orsuch CRE-positive cells are in particular useful for site-and timespecific gene targeting, for example in mouse model systems or ES-cellsystems; see, inter alia, Metzger, Methods 24 (2001), 71-80; Liu, Nat.Genet. 30 (2002), 66-72 or Yeh, PNAS 15 (2002), 13498-13503.

The recombinant nucleic acid molecule described and employed in thisinvention may also be a nucleic acid molecule comprising the DCXregulatory sequences described herein and nucleotide sequence to beexpressed under its control, whereby said expression-controlled sequenceis an antisense sequence, or encodes for a ribozyme or an inhibitingRNA-molecule.

The antisense sequences and ribozymes are molecules, the expression ofwhich occurs on the RNA level. “Antisense sequences” are sequences whichare complementary to an mRNA present in the target cell or a partthereof, the part possibly comprising the coding region, 5′- and/or3′-non-translated region. Antisense-RNAs, that is to say the transcriptsof the antisense sequence, are capable of hybridizing in vivo to thecomplementary mRNA and thereby to inhibit its translation. “Ribozymes”are catalytic RNA molecules. In context of the present invention theribozymes are preferably those which can bind specifically to an mRNA soas to render it inaccessible to successful translation by exerting acatalytic activity, preferably by hydrolytic cleavage. Instructions forselecting suitable antisense sequences and for constructing ribozymeswith the desired sequence specificity are described in the literatureand can be found for instance in “Antisense: From Technology to Therapy”(Schlingensiepen, R., Brysch, W., Schlingensiepen, K.-H., eds.,Blackwell Science Ltd. Oxford, 1997) or Rossi (AIDS Research and HumanRetroviruses 8 (1992), 183). Said ribozymes may also target DNAmolecules encoding the corresponding RNAs. Ribozymes are catalyticallyactive RNA molecules capable of cleaving RNA molecules and specifictarget sequences. By means of recombinant DNA techniques it is possibleto alter the specificity of ribozymes. There are various classes ofribozymes. For practical applications aiming at the specific cleavage ofthe transcript of a certain gene, use is preferably made ofrepresentatives of two different groups of ribozymes. The first group ismade up of ribozymes which belong to the group I intron ribozyme type.The second group consists of ribozymes which as a characteristicstructural feature exhibit the so-called “hammerhead” motif. Thespecific recognition of the target RNA molecule may be modified byaltering the sequences flanking this motif. By base pairing withsequences in the target molecule these sequences determine the positionat which the catalytic reaction and therefore the cleavage of the targetmolecule takes place. Since the sequence requirements for an efficientcleavage are low, it is in principle possible to develop specificribozymes for practically each desired RNA molecule. In order to produceDNA molecules encoding a ribozyme which specifically cleaves transcriptsof a gene encoding a protein to be inhibited oder inactivated, a DNAsequence encoding a catalytic domain of a ribozyme is bilaterally linkedwith DNA sequences which are homologous to sequences encoding the targetprotein. In accordance with this invention said sequence encoding aribozyme or its catalytic domain is under the control of the regulatorysequences (or functional parts or fragments thereof) of the invention.The expression of ribozymes in order to decrease the activity in certainproteins is also known to the person skilled in the art and is, forexample, described in EP-B1 0 321 201 or EP-B1 0 360 257.

Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, whereby the inhibitoryeffect is based on specific binding of a nucleic acid molecule to DNA orRNA. The antisense oligonucleotide of, e.g., at least 10 nucleotides inlength may be under the control of the regulatory sequences describedherein. The antisense DNA or RNA oligonucleotide hybridises to thedesired mRNA (which should be inactivated or inhibited) in vivo andblocks translation of said mRNA and/or leads to destabilization of themRNA molecule (Okano, J. Neurochem. 56 (1991), 560;Oligodeoxynucleotides as antisense inhibitors of gene expression, CRCPress, Boca Raton, Fla., USA (1988)).

For applying a triple-helix approach, a DNA oligonucleotide can bedesigned to be complementary to a region of the gene to be inhibited orinactivated according to the principles laid down in the prior art (seefor example Lee, Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241(1988), 456 and Dervan, Science 251 (1991), 1360). Such a triple helixforming oligonucleotide can then be used to prevent transcription of thespecific gene. The oligonucleotides described above can be delivered,inter alia, via a gene delivery vector as described below. Also thisapproach leads to in vivo inhibition of gene expression of therespective protein. The corresponding oligonucleotides have a length ofpreferably at least 10, in particular at least 15, and particularlypreferably of at least 50 nucleotides. They are characterized in thatthey specifically hybridize to said polynucleotide, that is to say thatthey do not or only to a very minor extent hybridize to other nucleicacid sequences.

A particularly preferred embodiment relates to the use of therecombinant nucleic acid molecules or vectors, wherein the antisensesequence, RNAi or the ribozyme is specific for to an inhibition and/ordown-regulation of an apoptosis-protein, like a caspase. This is ofparticular interest in context of this invention, since apoptotic andneurogenic regions in the adult brain overlap; see Biebl, Neurosci Left291 (2000), 17-20. Promoting the survival of neuronal determined cellsby means and methods provided herein raises the total population ofneuronal determined cells and neurons in nervous tissue, in in vitrocultures or in the adult brain. For example, the inhibition of caspase 3by the recombinant molecule described above is considered to lead toreduced cell death of neuronal determined cells in vivo and in vitro.

Preferred inhibiting RNA molecules (RNAi/iRNA) may be selected from thegroup consisting of RNAi, siRNA, shRNA and stRNA.

The term RNA interference (RNAi) is very well known in the art andusually describes the use of double-stranded RNA to target specificmRNAs for degradation, thereby silencing their expression.Double-stranded RNA (dsRNA) matching a gene sequence is synthesized invitro and introduced into a cell. The dsRNA feeds into a natural, butonly partially understood process including the highly conservednuclease dicer which cleaves dsRNA precursor molecules into shortinterfering RNAs (siRNAs). The generation and preparation of siRNA(s) aswell as the method for inhibiting the expression of a target gene is,inter alia, described in WO 02/055693, Wei, Dev. Biol. 15 (2000),239-255; La Count, Biochem. Paras. 111 (2000), 67-76; Baker, Curr. Biol.10 (2000), 1071-1074; Svoboda, Development 127 (2000), 4147-4156 orMarie, Curr. Biol. 10 (2000), 289-292. These siRNAs built then thesequence specific part of an RNA-induced silencing complex (RISC), amulticomplex nuclease that destroys messenger RNAs homologous to thesilencing trigger). Elbashir, EMBO J. 20 (2001), 6877-6888 showed thatduplexes of 21 nucleotide RNAs may be used in cell culture to interferewith gene expression in mammalian cells. It is already known that RNAiis mediated very efficiently by siRNA in mammalian cells but thegeneration of stable cell lines or non-human transgenic animals waslimited. However, new generations of vectors may be employed in order tostably express, e.g. short hairpin RNAs (shRNAs). Stable Expression ofShort Interfering RNAs in Mammalian Cells is inter alia shown inBrummelkamp, Science 296 (2002), 550-553. Also Paul, Nat. Biotechnol. 20(2002), 505-508 documented the effective expression of small interferingRNA in human cells. RNA interference by expression of short-interferingRNAs and hairpin RNAs in mammalian cells was also shown by Yu, Proc.Natl. Acad. Sci. U.S.A 99 (2002), 6047-6052. The shRNA approach for genesilencing is well known in the art and may comprise the use of st (smalltemporal) RNAs; see, inter alia, Paddison, Genes Dev. 16 (2002),948-958.

As mentioned above, approaches for gene silencing are known in the artand comprise “RNA”-approaches like RNAi or siRNA. Successful use of suchapproaches has been shown in Paddison (2002), loc. cit., Elbashir,Methods 26 (2002), 199-213; Novina, Mat. Med. Jun. 3 (2002), 2002;Donze, Nucl. Acids Res. 30 (2002), e46; Paul, Nat. Biotech 20 (2002),505-508; Lee, Nat. Biotech. 20 (2002), 500-505; Miyagashi, Nat. Biotech.20 (2002), 497-500; Yu, PNAS 99 (2002), 6047-6052 or Brummelkamp,Science 296 (2002), 550-553. These approaches may be vector-based, e.g.the pSUPER vector, or RNA polIII vectors may be employed as illustrated,inter alia, in Yu (2002), loc. cit.; Miyagishi (2002), loc. cit. orBrummelkamp (2002), loc. cit. It is envisaged that the regulatorysequences of the present invention are used in similar fashion as thesystems based on pSUPER or RNA polIII vectors.

Another preferred embodiment of the invention relates to the abovedescribed uses of nucleotide sequences comprising a fragment having alength of at least 15 nucleotides which specifically hybridizes understringent conditions to a strand of a DCX regulatory sequence describedin context of the invention.

Hybridizing nucleotide sequences according to the present embodiment canfor instance serve as probes which for instance contribute to identifyhomologous promoters, preferably regulatory sequences of other geneswhich, on account of certain corresponding sequence elements, induce anexpression pattern comparable to that of the regulatory sequences of theinvention. Moreover, these sequences can be used to design suitableoligonucleotides, for instance as PCR primers.

The term “hybridization” has already been defined further above. Thenucleotide sequences preferably hybridize under stringent conditions.The fragments have a length of at least 15 nucleotides, preferably of atleast 20 nucleotides, particularly preferably of at least 50nucleotides, especially preferably of at least 100 nucleotides,advantageously of at least 200 nucleotides and most preferably of atleast 500 nucleotides.

The invention also provides for the use of a vector comprising theinventive regulatory sequence or the recombinant nucleic acid moleculedescribed herein above, whereby said vector is preferably used for theearly transient expression of heterologous nucleotide sequences inproliferative neuronal determined cells.

The term “vector” relates to circular or linear nucleic acid moleculeswhich can autonomously replicate in host cells into which they areintroduced. The vectors may contain the above-characterized recombinantnucleic acid molecules in their full length or may contain, apart fromthe regulatory sequences of the invention, the components described forthe recombinant nucleic acid molecules, such as minimal promoter,polylinker and/or termination signal.

The vectors of the invention may be suitable for replication inprokaryotic and/or eukaryotic host cells. They contain a correspondingorigin of replication. The vectors are preferably suitable forreplication in mammalian cells, particularly preferably in human cells.

The vectors of the invention preferably contain a selection marker.Examples of selection marker genes are known to a skilled person.Selection marker genes which are suitable for selection in eukaryotichost cells are for instance genes for dihydrofolate reductase, G418 orneomycin resistance.

The vectors of the invention are preferably expression vectors forexpression in eukaryotic cells. Such vectors can be constructed startingfrom known expression vectors by replacement of their promoter or thesequences not belonging to a minimal promoter with the regulatorysequences of the invention or by supplementation with regulatorysequences (regulatory elements) of this invention. Examples ofexpression vectors which can be modified in this way are pcDVI(Pharmacia), pRC/CMV, pcDNA1 or pcDNA3 (Invitrogen).

As document in the appended examples, the vector of the presentinvention may, inter alia, comprise (a) marker or receptor gene(s) whichis GFP, EGFP or RFP.

In particular preferred embodiment of the invention the vector to beused comprises a nucleotide sequence selected from the group consistingof

-   -   (a) a nucleotide sequence as shown in SEQ ID NO: 5, 6 or 26 or        as shown in SEQ ID NO: 7, 8 or 27;    -   (b) a nucleotide sequence coding for the regulatory sequence and        the heterologous gene as comprised in the construct deposited        under accession number DSM 15111; and    -   (c) a nucleotide sequence which is at least 80%, more preferably        at least 85%, more preferably at least 90% and most preferably        at least 95% identical to the nucleotide sequence as shown in        SEQ ID NOS: 5 to 8, 26 or 27 and which comprises a regulatory        sequence of claim 1 or 2.

The corresponding sequences (SEQ ID NOs 5 to 8, 26 or 27) are alsoillustrated in the appended figures, examples or in the sequencelisting. Sequence identities of the vectors described herein may bededuced by methods known in the art and also illustrated herein above.

It is of note that the vectors to be employed in accordance with theinvention may also comprise functional parts or fragments of theregulatory sequence as disclosed herein. Functional fragments and partsmay also be deduced by methods described above and comprise, e.g.transfection studies with partial/fragmented regulatory sequenceslinked, inter alia to expressible marker/reporter genes.

In a particularly preferred embodiment, the above-described vectors areviruses.

In the state of art, a great number of viral vectors for transfection ofmammalian cells ex vivo or in vivo is described. These are derivativesof mammalian or human pathogenic viruses, which have been deprived oftheir pathogenic properties by genetic modification. For transfection,viral vectors are packaged in vitro according to methods known to askilled person, i.e. are provided with viral envelope proteins. DNA andRNA viruses can be used. Examples of viruses for transfection ofmammalian, preferably human cells, are Herpes virus, lentivirus,retroviruses, adenoviruses and adeno-associated viruses.

In another embodiment of the invention, the above-described vectors aresuitable for gene therapy or vaccination with a nucleic acid. Genetherapy and nucleic acid vaccination are based on the introduction oftherapeutic or immunizing genes into cells ex vivo or in vivo. Suitablevectors or vector systems and methods for using them for gene therapy orDNA/RNA vaccination are described in the literature and are known to askilled person, see for instance Giordano, Nature Medicine 2 (1996),534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256(1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957; Schaper, Current Opinion in Biotechnology 7(1996), 635-640; or Verma, Nature 389 (1997), 239-242 Geddes, FrontNeuroendocrinol. 20 (1999), 296-316 and the references cited therein. Ofparticular interest is the fact that gene delivery systems/gene therapyapproaches in neurology have also been described by Tuszyski, J.Neurosci. 19 (2002), 207 or Blesch, Brain Res Bull. 57 (2002), 833-833.Global gene and cell replacement strategies via stem cells and the likehave been proposed and described by Park, Gene Therapy 9 (2002),613-624. The above-described recombinant nucleic acid molecules orvectors can for instance be designed for the direct introduction or forintroduction via liposomes or viral vectors, e.g. adenoviral,lentiviral, adeno-associated viral (AAV), herpes vectors or retroviralvectors.

In a further preferred embodiment, the invention relates to agenetically modified cell/host cell which comprises the regulatorysequence, the recombinant nucleic acid molecule or the vector asdescribed above. Also provided is/are (a) method(s) for preparinggenetically modified host cells, characterized in that the host cellsare transfected with one of the above-described vectors and thetransfected host cell is cultured in a culture medium.

The term “genetically modified” means that the host cell or the hostcontains, in addition to the natural genome, a nucleic acid molecule ora vector of the present invention, which has been introduced into thehost cell or the host or into a precursor. The nucleic acid molecule orthe vector can be present in the genetically modified host cell/hosteither as an independent molecule outside the genome, preferably as areplicable molecule, or may be stably integrated in the genome of thehost cell or host.

The introduction of a vector into host cells can be carried outaccording to known standard methods as for instance described inSambrook (loc.cit.). Examples of applicable transfection techniques arecalcium phosphate transfection, DEAE dextran-mediated transfection,electroporation, transduction, infection, lipofection or biolistictransfer. Subsequent culturing can be carried out using standard methodstoo, or in the case of the genetic modification of neuronal cells,preferably the methods described in the Examples and the referencescited therein.

In another preferred embodiment, the invention relates to host cellswhich are genetically modified with a regulatory sequence, a recombinantnucleic acid molecule or a vector of the present invention or areobtainable by the above-described method. Most preferably the host cellis capable of proliferation and is able to acquire (for example underexperimental in vivo or in vitro conditions) a neuronal phenotype.

The host cell of the present invention can in principle be anyprokaryotic or eukaryotic cell and includes, inter alia, mammaliancells, fungal cells, plant cells, insect cells or bacterial cells.

The host cells of the present invention should be able to be mitoticallyactive and should be able to acquire a neural phenotype, in particularunder in vitro conditions. Suitable bacterial cells are those which aregenerally used for cloning, such as E. coli or Bacillus subtilis.Examples of fungal cells are yeast cells, preferably those of the generaSaccharomyces or Pichia, particularly preferably of Saccharomycescerevisiae or Pichia pastoris. Suitable animal cells include forinstance insect cells, vertebrate cells, preferably mammalian cells,such as CHO, COS7, Hela, NIH3T3, MOLT4, Jurkat, K562, HepG2, PC12, Neuro2A, P19 teratocarcinoma and the like. Yet, also cultured primary cellsare envisaged, like cultured hippocampal cells or cultured RPE-cells.Further suitable cell lines are described in the art and can forinstance be obtained from the Deutsche Sammlung für Mikroorganismen undZellkulturen (DSMZ, Braunschweig). In this context it is of note thatalso non-neuronal cells may be transfected with a regulatory sequence, arecombinant nucleic acid sequence or a vector of the invention. Thesetransfected cells are particularly useful in methods described hereinbelow relating, inter alia, to screening methods for compounds capableof inducing differentiation/determination programs in cells which lead,either directly or indirectly, to a neuron or a neuron-like phenotype.As illustrated in the appended examples, a non-stimulated COS7 cell,transfected with a construct described herein, i.e. an EGFP-gene underthe control of the regulatory sequence of the present invention, does,under normal culturing conditions, not express said marker/reporter.Yet, these cells may be employed in screening systems, for examplehigh-throughput screenings, were compounds are tested for their capacityto activate the regulatory sequence of the invention.

The embodiment of the host cells which are brain or neuronal cells isparticularly preferred.

Neuronal progenitor or stem cells as well as general embryonic stemcells (ES cells) are the primary site of application of the regulatorysequences, recombinant nucleic acid molecules or vectors of theinvention. In particular embryonic stem cells transfected with theregulatory sequences, recombinant nucleic acid molecules or vectors areuseful in methods described herein below and in high-throughputscreenings. The isolated cells can also be precursor or a stem cellswhich can be converted into neuronal determined cells by suitable invitro culturing. Corresponding methods are described in the art. Hostcells of mammalian, most preferably human origin are particularlypreferred in the present invention.

Accordingly, the host cell of the invention may be a neuronal cell, anES-cell, a germ cell (for example a zygote which is particularly usefulfor the preparation of non-human transgenic animals as detailed in theappended examples) or, a cultured cell line as defined above or aprimary cell, like hippocampal cells, RPE-cells or olfactory bulb cells.

The invention also relates to a method for preparing a geneticallymodified host cell, characterized in that the host cell is transfectedwith a nucleic acid molecule which is or which comprises the DCXregulatory sequence, the recombinant nucleic acid molecule or the vectordescribed above.

Various methods are known in the art for introducing nucleic acidmolecules into host cells. These include but are not limited tomicroinjection, in which DNA is injected directly into the nucleus ofcells through fine glass needles; dextran incubation, in which DNA isincubated with an inert carbohydrate polymer (dextran) to which apositively charged chemical group (DEAE, for diethylaminoethyl) has beencoupled. The DNA sticks to the DEAE-dextran via its negatively chargedphosphate groups. These large DNA-containing particles stick in turn tothe surfaces of cells, which are thought to take them in by a processknown as endocytosis. Some of the DNA evades destruction in thecytoplasm of the cell and escapes to the nucleus, where it can betranscribed into RNA like any other gene in the cell; calcium phosphatecoprecipitation, in which cells efficiently take in DNA in the form of aprecipitate with calcium phosphate; electroporation, in which cells areplaced in a solution containing DNA and subjected to a brief electricalpulse that causes holes to open transiently in their membranes. DNAenters through the holes directly into the cytoplasm, bypassing theendocytic vesicles through which they pass in the DEAE-dextran andcalcium phosphate procedures (passage through these vesicles maysometimes destroy or damage DNA), liposomal mediated transformation, inwhich. DNA is incorporated into artificial lipid vesicles, liposomes,which fuse with the cell membrane, delivering their contents directlyinto the cytoplasm; biolistic transformation, in which DNA is absorbedto the surface of gold particles and fired into cells under highpressure using a ballistic device; and viral-mediated transformation, inwhich nucleic acid molecules are introduced into cells using viralvectors. Since viral growth depends on the ability to get the viralgenome into cells, viruses have devised efficient methods for doing so.These viruses include, as mentioned above, retroviruses and lentivirus,adenovirus, herpesvirus, and adeno-associated virus. As indicated, someof these methods of transforming a cell require the use of anintermediate plasmid vector. U.S. Pat. No. 4,237,224 to Cohen and Boyerdescribes the production of expression systems in the form ofrecombinant plasmids using restriction enzyme cleavage and ligation withDNA ligase. These recombinant plasmids are then introduced by means oftransformation and replicated in unicellular cultures includingprokaryotic organisms and eukaryotic cells grown in tissue culture. TheDNA sequences are cloned into the plasmid vector using standard cloningprocedures known in the art, as described by Sambrook (1989), loc.cit.In accordance with one of the below-described methods, a nucleic acidmolecule encoding, e.g. GFP under the control of a regulatory sequenceof the present invention is thus introduced into a plurality of cells.The regulatory sequence which controls expression of the GFP, however,only functions in the cell type of interest (i.e. neuronal determinedcells). Therefore, the GFP is only expressed in the cell type ofinterest. Since GFP is a fluorescent protein, the cells of interest cantherefore be identified from among the plurality of cells by thefluorescence of the GFP.

Any suitable means of detecting the fluorescent cells can be used. Thecells may be identified using epifluorescence optics, and can bephysically picked up and isolated by mechanical devices such as Quixell(Stoelting, Inc., St. Louis, Mo.) or Laser Tweezers (Cell Robotics Inc.,Albuquerque, N. Mex.). They can also be separated in bulk throughfluorescence-activated cell sorting, a method that effectively separatesthe fluorescent cells from the non-fluorescent cells (e.g., Wang(1998)).

As will be further explained below, one embodiment of the presentinvention thus provides for the isolation and enrichment of neuronaldetermined cells from embryonic and adult nervous tissue, in particularbrain of both rodent and human derivation. Specifically,fluorescence-activated cell sorting of human hippocampal cells,transfected with the fluorescent protein of choice driven by the DCXpromoter/enhancer described herein is provided.

As also illustrated in the appended examples, one of the basic findingsof the invention is the fact that herein generated non-human transgenicanimals express heterologous nucleic acid sequences under the control ofthe regulatory sequences provided herein and that said expression islimited to cells which are neuronal determined/restricted andmitotically active.

Therefore, the present invention also provides a non-human transgenicanimal which comprises host cells of the present invention and/or whichcomprises in its cells at least one additional copy of the regulatorysequence of the invention or which comprises in its cells a recombinantnucleic acid molecule of the invention. As shown in the examples, thegeneration of such a transgenic animal is within the skill of a skilledartisan. Corresponding techniques are, inter alia, described in “CurrentProtocols in Neuroscience” (2001), John Wiley & Sons, Chapter 3.16.Accordingly, the invention also relates to a method for the generationof a non-human transgenic animal comprising the step of introducing aregulatory sequence or a recombinant nucleic acid molecule of theinvention into an ES-cell or a germ cell. The transgenic non-humananimal may be selected from a plurality of transgenic animals e.g.non-vertebrates like C. elegans, Drosophila or vertebrates, likechicken. Yet, more preferred is a non-human transgenic which is amammal, more preferably a rat or a pig, and most preferably mouse.

Preferably, the non-human transgenic animal comprises in its cells arecombinant nucleic acid molecule expressing a marker gene under thecontrol of the regulatory sequence of this invention. Most preferably,the non-human transgenic animal comprises in its cells a recombinantnucleic acid molecule comprising the regulatory sequence of theinvention and additionally a nucleotide sequence under the control ofsaid regulatory sequence and encoding a marker/reporter gene, like greenfluorescent protein (GFP or EGFP). Further corresponding embodiments arein the Examples. The non-human transgenic animal provided and describedherein is particular useful in screening methods and pharmacologicaltests described herein below. In particular the non-human transgenicanimal described herein may be employed in drug screening assays as wellas in scientific and medical studies wherein neuronal determined cellsand/or are tracked, selected and/or isolated.

In a further embodiment the invention provides for a compositioncomprising the regulatory sequence, the recombinant nucleic acidmolecule, the vector or a genetically modified host cell describedherein. Said composition may also comprise cells or tissue derivedand/or obtained from a transgenic animal described above. Such cells ortissue may be particularly useful in xenografts. Most preferably, saidcomposition is a pharmaceutical composition, optionally comprising apharmaceutically acceptable carrier or diluent or a diagnosticcomposition, optionally further comprising suitable means of detection.

For formulating cells for administration as a pharmaceuticalcomposition, the cells are suspended in a pharmaceutically acceptablecarrier material. This applies, inter alia to neuronal determined cellsobtained or selected by the methods described herein. Examples ofcarrier material are water, sodium chloride solution, dextrose, glyceroletc. or combinations thereof. In addition, the cell suspension to beadministered may contain further substances, such as emulsifying agents,pH buffer, adjuvants or also neurotrophic factors, such as BDNF or NT-3and the like.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal or intranasaladministration and the like. Particularly preferred is intra-cerebraladministration. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatients size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Proteinaceouspharmaceutically active matter may be present in amounts between 1 μgand 10 mg per dose; however, doses below or above this exemplary rangeare envisioned, especially considering the aforementioned factors. Ifthe regimen is a continuous infusion, it should also be in the range of1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Thecompositions of the invention may be administered locally orsystemically. The compositions of the invention may also be administereddirectly to the target site, e.g., by biolistic delivery to an internalor external target site or by catheter to a site in an artery.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents depending on the intended use ofthe pharmaceutical composition.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilized powder which may contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use. After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labeled for treatment of an indicated condition.Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of culturedneuronal cells, cell lines, or in animal models, usually mice, rabbits,dogs, or pigs. The animal model may also be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration. The exact dosage will be determined by the practitioner,in light of factors related to the subject that requires treatment.Dosage and administration are adjusted to provide sufficient levels ofthe active moiety or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

Modes of administering nucleic acid pharmaceuticals are described in theart, e.g. (Fynan, Proc. Natl. Acad. Sci. USA 90 (1993), 11478-11482;Boyer, Nat. Med. 3 (1997), 526-532; Webster, Vaccine 12 (1994),1495-1498; Montgomery, DNA Cell Biol. 12 (1993), 777-783; Barry, Nature311 (1995), 632-635; Xu and Liew, Immunology 84 (1995), 173-176; Zhoug,Eur. J. Immunol. 26 (1996), 2749-2757; Luke, J. Inf. Dis. 175 (1997),91-97; Mor, Biochem. Pharmacology 55 (1998), 1151-1153; Donelly, Annu.Rev. Immun. 15 (1997), 617-648; MacGregor, J. Infect. Dis. 178 (1998),92-100). Particular examples from the neurological field comprise Blits,Cell Transplant 11 (2002), 593-613; Boulis, J. Neurosurg. 96 (2002),212-219. As mentioned above, gene therapy approaches are currently underway for certain disorders and offer novel treatment methods, see, interalia, Frank, Surg. Oncol. Clin. N. Am. 11 (2002), 589-606 or Hull, Nurs.Stand. 17 (2002), 3942.

For use the nucleic acid molecules described herein can be formulated ina neutral form or as a salt. Pharmaceutically effective salts are knownto a skilled person. The nucleic acid molecules, in particular therecombinant nucleic acid molecules described above can be used interalia to treat and/or to prevent neurological disorders and areadministered in doses which are pharmacologically effective forprophylaxis or treatment.

Pharmaceutical compositions for injection are typically prepared as aliquid solution or suspension. The preparations can be emulsified or theactive ingredient can be encapsulated in liposomes. The activeingredients are often mixed with carrier materials which are compatiblewith the active ingredient. Examples of carrier materials are water,sodium chloride solution, dextrose, glycerol, ethanol etc orcombinations thereof. The vaccine as well as other nucleic acidpharmaceuticals of the invention may also contain auxiliary substances,such as emulsifiers, pH buffers and/or adjuvants.

DNA can be administered by biolistic transfer instead of by injection(U.S. Pat. No. 5,100,702; Kalkbrenner, Meth. Mol. Biol. 83 (1996),203-216). For this purpose, DNA, that is to say recombinant nucleic acidmolecules or vectors of the present invention, are bound to smallparticles, for instance gold particles or particles of biocompatiblematerial, and, accelerated by gas pressure, are introduced into thebrain. DNA can also be administered orally or sublingually or applied tothe mucosa of the respiratory tract by nasal or intratrachealapplication. (for this, examples are given in Etchart, J. Gen. Virol. 78(1997), 1577-1580 or McCluskie, Antisense and Nucleic Acid DrugDevelopment 8 (1998), 401-414).

Of particular interest in context of this invention is also apharmaceutical composition comprising proliferative neuronaldetermined/restricted cells as isolated and/or obtained by methodsdescribed herein below.

A “patient” or “subject” for the purposes of the present inventionincludes both humans and other animals, particularly mammals, and otherorganisms. Thus, the methods are applicable to both human therapy andveterinary applications. In the preferred embodiment the patient is amammal, and in the most preferred embodiment the patient is human. Mostpreferably, the pharmaceutical composition to be prepared in accordancewith this invention is to be administered by one or several of thefollowing modes: Administration can be oral, intravenous, intraarterial,intratracheal, intranasal, subcutaneous, intramuscular, intracranial(i.e. intraventricular), intra-cerebral or intraspinal (intrathecal),epidermal or transdermal, pulmonary (e.g. inhalation or insufflation ofaerosol or powder), by delivery to the oral or rectal mucosa as well asophthalmic delivery.

In another embodiment, the present invention provides for a kitcomprising the regulatory sequence, the recombinant nucleic acidmolecule, the vector of any one or a genetically modified host celldescribed herein.

The invention furthermore provides for the use of a recombinant nucleicacid molecule, of a vector or of a genetically modified host cell (allexpressing or capable of expression of a heterologous nucleic acidmolecule under the control of the DCX promoter described herein) for thepreparation of a pharmaceutical composition for the treatment of aneurological disorder or disease. Said neurological disorder or diseaseis preferably a neurodegenerative disease, an injury of the CNS or PNS,hypoxia, ischemia, epilepsy, stroke, CNS trauma, a tumorous disorder ofthe nervous system, a neural disorder caused by toxicological insult, aneuro-ophthal mological disorder, a psychiatric disorder, an age-relatedneurological loss or damage, or is neurological disorder caused by adevelopmental malformation, a brain malformation or a neural migrationdisorder. Neurodegenerative diseases in this context are, inter alia,Parkinson's disease, Alzheimer's disease, ALS, Creutzfeld-Jacobs diseaseor dementia, like HIV-related dementia. As mentioned above, also otherneurological disorders, like Huntingston's disease, stroke, ischemia,injuries, like spinal cord injuries or brain injuries, or toxic insultsmay be treated with the compounds of the present invention.

It is also envisaged that the recombinant nucleic acid molecule, thevector or the genetically modified host cell as described herein andcapable of expressing a heterologous nucleic acid sequence is employedin the preparation of a pharmaceutical composition for the treatment oflearning and/or memory disorders or for the enhancement of memory orlearning skills. Memory or learning disorder comprise, but are notlimited to, dementias, traumas, syphilis or apraxias. It is envisagedthat, for example a recombinant nucleic acid molecule as describedherein and consisting of the DCX promoter (or a functional fragmentthereof) and a heterologous gene (driven by said promoter and expressingan anti-apoptotic gene is employed.

Accordingly, the present invention also provides for a method fortreating and/or preventing a neurological disorder or disease comprisingthe administration of a compound of the present invention to a subjectin need of such a treatment. Preferably said subject is a human patient.As will be discussed herein below, the present invention also providesfor means and methods for the detection and/or isolation of neuronaldetermined/restricted cells. These cells are also particularly useful inmedical settings and may also be comprised in pharmaceuticalcompositions of the invention and be employed in treatment and/orprevention regimes on, preferably, human patients.

Therefore, and as illustrated herein below, the invention also providesfor the use of a recombinant nucleic acid molecule or of a vector of theinvention for the preparation of a diagnostic composition for thedetection of (a) neuronal determined cell(s). Furthermore, the use of arecombinant nucleic acid, of a vector or a genetically modified hostcell described herein for the preparation of a diagnostic compositionfor the isolation of (a) proliferating neuronal determined cell cell(s)is an embodiment of the present invention.

In yet a further embodiment, the invention relates to a method for thedetection of (a) neuronal proliferating determined cell(s) comprisingthe steps of (a) expressing in a plurality of cells a recombinantnucleic acid or a vector of the invention; and (b) selecting cells whichexpress a heterologous nucleotide sequence under the control of theregulatory sequence as defined herein. In one embodiment said methodcomprises in step (b) the detection of an expressed, heterologous markeror reporter gene. Said method may comprise the detection of an expressedfluorescent marker, like GFP and derivatives thereof or gene coding foran enzyme, like β-Gal, CAT or luciferase. As illustrated herein below,the methods provided herein may comprises a FACS (Fluorescence activatedcell sorting) analysis, MACS (Magnetic cell sorting via, inter alia, MSseparation columns) analysis or affinity isolation (for example withmagnetic beads and the like) of cells to be selected and isolated. Suchmethods are known by the person skilled in the art, see, e.g. Kawaguchi,Mol. Cell Neurosci. 17 (2001), 259-273; WO 00/23571 or WO 01/53503.

As pointed out herein above, the present invention provides for thefirst time means for the detection and isolation of proliferative,neuronal-determined/restricted cells in vivo. Since DCX is anintracellularly expressed protein, selection systems based on detectionof expressed DCX-mRNA or DCX-protein may only be carried out when cellsare fixed and/or permeabilized and, accordingly, destroyed. Yet, thepresent invention provides for detection/selection systems allowing foran in vivo detection/selection of proliferative neuronalrestricted/determined cells, in particular neuronalrestricted/determined stem or progenitor cells. Due to the provision ofspecific DCX-regulatory sequences the invention, therefore, allows forunique and simple isolation approaches for desired neuronal cells, i.e.neuronal restricted cells which do not develop into undesired cells,like, e.g. astrocytes, oligodendrocytes or even non-neural cells, likecells of the hematopoetic system or epithelial cells. Accordingly, theinvention also provides for a method for the detection of (a)proliferative neuronal restricted determined cell(s) comprising the stepof selecting in a plurality of cells (a) cell(s) which comprise(s) anactivated DCX regulatory sequence. Said method may additionally comprisea step of separating (a) cell(s) which express(es) a heterologousnucleotide sequence under the control of the regulatory sequencedescribed herein from (a) cell(s) which is/are not capable of expressingsaid heterologous nucleotide sequence or may comprise an additional stepof separating (a) cell(s) which comprise an activated regulatorysequence as defined herein. The activation of said regulatory sequencemay, inter alia, be determined by the expression of a gene or a nucleicacid sequence encoding for a marker or a reporter as defined above. Saidmarker/reporter may, e.g. comprise a fluorescent protein or an enzyme,like luciferase or β-gal and the like.

Accordingly, the invention also provides for a method of separatingproliferative neuronal determined cells from a mixed population of celltypes from nervous tissue, based upon cell type-selective expression ofthe specific regulatory sequences/“promoters” for DCX. This methodincludes selecting the DCX regulatory sequence as defined herein whichfunctions selectively in the neuronal determined cells, introducing anucleic acid molecule encoding a marker, marker protein like afluorescent protein under control of said promoter into all cell typesof the mixed population of cell types from nervous tissue, e.g.hippocampal tissue allowing only the neuronal determined cells, but notother cell types, within the mixed population to express said markerprotein, identifying cells of the mixed population of cell types thatexpress said marker/marker protein, which are restricted to neuronaldetermined cells, and separating the marked/labelled cells from themixed population of cell types, wherein the separated cells arerestricted to neuronal determined cells. The person skilled in art isreadily in a position to carry out the methods provided herein. Methodsfor separating cells are described in WO 98/32879, WO 00/23571 or WO01/53503.

Due to the methods provided herein, it is now possible to obtain adistinct population of proliferative neuronal determined/restrictedcells. Accordingly, in one embodiment, the present invention relates toan isolated neuronal determined cell or an enriched or purifiedpreparation of isolated neuronal determined cells and progeny thereof.In context of the embodiments provided herein, the term “isolated”relates to (a) proliferative neuronal determined cell(s) which areobtained by methods described herein and have been separated from(nervous) tissue and/or cell populations/suspensions which compriseother neural cells, like glial cells, microglia cells, endothelial cellsoligodendrocytes or mature, fully developed neurons and the like. Alsoenvisaged is the isolation and separation of (a) proliferative neuronaldetermined cell(s) from multi/pluripotent undifferentiated stem cells.

The distinct all population of proliferative neuronaldetermined/restricted cells as obtained by the methods of this inventionare particularly useful I screening assays (for example for neuroactivesubstances) as well as for medical purposes.

The invention relates to a detection of mitotically active neuronaldetermined/restricted cells or to an isolated and/or enriched orpurified preparation of isolated, proliferative neuronalrestricted/determined cells. The invention also relates to a method ofseparating proliferative neuronal restricted/determined cells from amixed population of cells from nervous tissue or other tissues of anorganism, such as chicken, rodent or human, based upon cell-typespecific expression of a neuronal restricted/determined cells specificpromoter/regulatory element, as disclosed herein. This inventioncomprises, e.g. the selective expression of a nucleic acid moleculeencoding a detectable marker (fluorescent protein, luminescent protein,surface antigen, tag, etc.) under the control of the DCX regulatorysequence described herein. When using a fluorescent protein, thefluorescent cells of a mixed population of cell types are mitoticallyactive neuronal determined/restricted cells and can be identified andseparated from the rest of the cells. The hereby separated cells areneuronal determined/restricted cells, as documented in the appendedexamples. The promoter/regulatory sequence described herein specificallydrives expression in neuronal determined/restricted cells, but not inother cells from the organism or from the mixed population of cells.

The regulatory sequence of the invention driving the expression of adetectable marker can be introduced into a plurality of cells, organsand organisms. This includes, but is not limited to, the introductioninto cells by various methods known to those of ordinary skill in theart, for example transfection (liposomal based, electroporation,ballistic based, etc.) or viral mediated transduction (adenoviral,retroviral, etc.), and the introduction into organisms by variousmethods known to those of ordinary skill in the art, for exampletransfection (liposomal based, electroporation, biolistic based), viralmediated transduction (adenoviral, retroviral) and transgenictechnology.

After cell specific expression of the detectable marker protein, e.g.green fluorescent protein (GFP) or enhanced green fluorescent protein(EGFP), the cells expressing the fluorescent protein are detected and/orseparated by appropriate means, such as fluorescent activated cellsorting. Methods for isolation and separating cells expressing adetectable marker are available and known by skilled persons (Roy, J.Neuroscience 59 (2000), 321-331; Kawaguchi 2001, loc.cit.). In spite ofthe embodiment described above, the invention also relates to the one ofherein described regulating sequences for the expression of otherheterologous nucleic acid sequences besides marker or reporter genes.

In another preferred embodiment, the present invention relates to theuse of the regulatory sequences or the recombinant nucleic acidmolecules or vectors of the invention, which preferably express areporter gene, for identifying and isolating cis-elements from theregulatory sequence which mediate neuronal determined/restrictedcell-specific expression.

In another preferred embodiment, the present invention relates to theuse of the regulatory sequences or the recombinant nucleic acidmolecules or vectors of the invention, which preferably express areporter gene, for determining the degree of maturation of neuronaldetermined/restricted cells or for determining the influence ofcandidate compounds/drugs and the like on their differentiation programand/or their development. This embodiment can be used for instance todetermine the degree of maturation of in vitro cultured neuronal cellsor neural stem cells which are to be used in clinical studies. Yet, itis also envisaged that the degree of maturation of other cells (humanand non-human cells) be assayed by the uses and methods provided herein.For example, it is envisaged that cells, e.g. non-neuronal/neural cells,like HEK293-cells, fibroblasts, are transfected/transduced withrecombinant nucleic acid molecules as described above and said cells areemployed in screening methods as provided herein. Accordingly, thesetransfected/transduced (host) cells may provide valuable tools in drugscreening assays. As documented in the appended examples expressiondriven by the regulatory sequences of doublecortin provides for uniquemeans to determine the developmental stage of a neuron-restricted cell,since doublecortin is merely transiently expressed during a restrictedearly phase during neuron development or differentiation.

Another embodiment of the invention relates to the use of the regulatorysequences, the recombinant nucleic acid molecules, the vectors, the hostcells or the transgenic non-human animals (or their cells, tissues ororgans) described herein for identifying and isolating factors whichmediate neuron-restricted/neuronal determined cell-specific expression.Detailed methods for such an use are described herein below.

As already mentioned above, the neuronal determined/restricted cellswhich may be detected and isolated by methods provided herein are alsouseful in medical settings and in the prevention and/or treatment ofneurological disorders and/or the treatment or prevention of learning ormemory disorders. Therefore, the invention relates, in one embodiment,to the use of (a) neuronal determined cell(s) detected or as isolated bythe method described herein for the preparation of a pharmaceuticalcomposition for the treatment of a neurological disorder or disease. Thepresent invention also provides means for the isolation and separationof proliferative neuronal restricted/determined cells from a mixedpopulation of cells or from organs or organisms of different species.These isolated cells can be used for different purposes, includingstudying the molecular and cellular properties of this cell population,and for medical transplantation. Transplantation strategies for thediseased central nervous system (CNS) require non-tumorogenic, wellcharacterized cells which are able to differentiate into the appropriatecell types. For neuronal cell replacement, neuronaldetermined/restricted cells are most suitable since they are alreadydetermined to become neurons, but are not yet fully differentiated andstill flexible enough to acquire the neuronal phenotype required.

The recombinant nucleic acid molecule or of a vector of the inventionmay also be used for determining the degree of maturation of a neuronalcell. The person skilled in the art may for example employ transfectionstudies wherein the recombinant nucleic acid molecule or of a vector ofthe invention drives the expression of a marker or reporter gene. Byanalyzing the degree of expression of said marker or reporter gene, theskilled artisan is readily in a position to determine the developmentalstate of a cell transfected or transduced with said recombinant nucleicacid molecule or said vector. A positive signal indicates that the cellto be analyzed has the neuronal determination/differentiation programswitched on, since the herein disclosed DCX regulatory sequence isactive.

The recombinant nucleic acid molecule, the vector, the geneticallymodified host cell or the non-human transgenic animal (or organs, tissueor cells thereof) of the present invention may also be employed and usedfor identifying and/or isolating factors and/or compounds capable ofregulating neuronal determined cell activity, neurogenesis and/orneuronal differentiation or migration. Accordingly, the recombinantnucleic acid molecule, the vector, the genetically modified host cell orthe non-human transgenic animal (or parts like isolated organs and cellsof said animal) of the present invention may, inter alia, be employed inscreening methods as described. Said recombinant nucleic acid molecule,said vector, and/or said genetically modified host cell of the inventionare particularly useful for the generation of a non-human transgenicanimal.

Therefore, the invention also provides for non-human transgenic animalsas well as to the use of said transgenic, non-human animal foridentifying and/or obtaining a molecule which is capable of modifyingthe cell fate of a neuronal stem cell or a neuronal progenitor.Preferably, said transgenic animal comprises in its somatic and/or germcells at least one additional copy of the DCX regulatory sequence (or afunctional part or a fragment thereof) which controls the expression ornon-expression of a selectable marker/reporter gene or of any furtherheterologous nucleic acid molecule. Such an animal may be a “knock-in”animal, expressing, e.g., a marker/reporter gene, like GFP, EGFP orDsRed, under the control of a regulatory sequence provided herein. Yet,an inventive transgenic animal may also be a transgenic animal whichdoes not express a nucleic acid sequence under the control of theregulatory sequence described herein. Such animal may be a transgenicanimal in which the corresponding, endogenous doublecortin (DCX)regulatory sequence has been inactivated or deleted (e.g. a “knock-outanimal” or an animal comprising a mutated, non-functional version of thecorresponding DCX regulatory sequences). In said “knock-out animal” theherein described regulatory sequences are inactivated or suppressed. Theappended examples illustrate how such transgenic animals may be obtainedand employed in the methods of the present invention.

A most preferred non-human transgenic animal of the present inventioncomprises in its cells at least one additional copy of the regulatorylinked sequence described herein, preferably operatively linked to aheterologous gene, like a gene coding for a marker/reporter (e.g.luciferase, GFP, DsRed, EGFP and the like).

In a particular preferred embodiment of the invention, a method forscreening of compounds capable of regulating neuronal determined cellactivity, neurogenesis and/or neuronal differentiation is disclosed,said method comprising the steps of: (a) contacting a recombinantnucleic acid molecule, a vector or a genetically modified host cell or anon-human transgenic animal of the invention or a non-human transgenicanimal as generated by the method of the invention with (a) compound(s)suspected to directly or indirectly interact with the regulatorysequence as defined herein; and (b) it is detected whether saidcompound(s) is/are capable of interacting with said regulatory sequence.

Said “contacting” may be carried out in vivo as well as in vitro. It is,e.g. envisaged that said transgenic animal is contacted in vivo with thecompound/candidates to be tested. Said compound(s) may, inter alia, beinjected to said animal, for example by inter-cerebral/inter-cranialinjection. Similarly, cells transfected with a recombinant nucleic acidmolecule as disclosed herein may be contacted in vitro with thecompounds to be tested, for example by introducing the test compoundsinto the culture medium. The detection, whether said compound(s) is/arecapable of interacting with regulatory sequence of the invention, mayinvolve the detection, whether the regulatory sequence is activated.Corresponding, non limiting models are given herein below and in theappended examples.

The term “contacting a recombinant nucleic acid molecule of theinvention with (a) compound(s) suspected to directly or indirectlyinteract said the regulatory sequence” may comprise tests ofinteraction. Such tests may be carried out by specific immunological,biochemical assays and/or genetic assays which are known in the art andcomprise homogenous and heterogeneous assays. For example, in the methodof the present invention, the interaction assays to be employed inaccordance with this invention may be used to detect as a response thedirect or indirect interaction of the regulatory sequence with thecandidate molecule. Said interaction assays employing read-out systemsare well known in the art and comprise, inter alia, two hybridscreenings, (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO00/02911 and modified for detection of interaction partners for theregulatory sequences of the invention), GST-pull-down columns,co-precipitation assays from cell extracts as described, inter alia, inKasus-Jacobi, Oncogene 19 (2000), 2052-2059, “interaction-trap” systems(as described, inter alia, in U.S. Pat. No. 6,004,746), in vitro bindingassays and the like. Further interaction assay methods and correspondingread out systems are, inter alia, described in U.S. Pat. No. 5,525,490,WO 99/51741, WO 06/17221, WO 00/14271, WO 00/05410. Of particularrelevance in accordance with this invention are interaction assays whichcomprise biochemical/genetic methods like, e.g. band shift assays whichare employed to deduce, inter alia, proteineous compounds capable ofinteracting with regulatory sequences/promoters.

Furthermore, the above recited method for screening of compounds capableof regulating neuronal determined cell activity, neurogenesis and/orneuronal differentiation may comprise the screening for substances whichare capable to induce a differentiation program in a test/host cellcomprising the regulatory sequence of the present invention. Saidscreening methods may also comprise the screening of compounds whichactivate, directly or indirectly, the regulatory sequence of DCX asdefined herein and lead to a migration of a cell wherein said regulatorysequence is activated. The present invention provides for drug screeningmethods, as will be detailed below.

A difference in the expression profile of a nucleic acid molecule underthe control of a regulatory sequence (or a functional part of fragmentthereof) in the absence of the candidate agent as compared with theexpression profile in the presence of the candidate agent indicates thatthe agent modulates the expression of DCX. Said agent is, accordingly,capable of interacting with the regulatory sequence” of the presentinvention, when a corresponding readout scores positively. For example,a candidate agent may provoke the expression of a marker or reportergene, like EGFP, DsRed or GFP under the control the herein describedDCX-regulatory sequence. In this case, the candidate compound has,either directly or indirectly, interacted with said regulatory sequence.A direct interaction is, inter alia, envisaged from a specifictranscription factor capable of binding to the regulatory sequence ofthe invention and of eliciting the transcription. An example of indirectinteraction of the candidate compound comprises, but does not limitedto, the involvement of a signal transduction pathway. Similarly, it isalso envisaged that difference in the physiological response, forexample of the electrophysiological response, in absence or in presenceof the candidate molecule to be tested for specific modulation theactivity of the regulatory sequence of the present invention indicatesthat the candidate agent/molecule/compound is capable of modifying saidactivity. The difference, as used herein, is statistically significantand preferably represents at least a 30%, more preferably at least 50%,more preferably at least a 90% difference. Accordingly, the DCX-specificpromoter disclosed herein may be linked to a suitable reporter gene,e.g. DsRed, luciferase or GFP and the like, and used in cell-basedassays to screen for compounds capable of modulating, via up- ordown-regulation of DCX, a molecule which is transiently expressed inneurogenesis, as detailed in the appended examples.

The above mentioned comparison between the response upon contacting theregulatory sequence of the invention with said candidate molecule andthe standard response as measured in the absence of said candidatemolecule may proived for the presence, the absence, the decrease or theincrease of a specific signal in the readout system. Said readoutsystem, as described herein may be a, e.g., a biochemical or aphysiological readout system, like a electrophysiological readoutsystem. Genetic readout systems are also envisaged. A specific signalwhich is increased over the standard signal/response may thereby beclassified as being an activator of the DCX regulatory sequence providedherein, whereas a decreased signal may be classified as being diagnosticfor an inhibitor of DCX regulatory sequence function or expression.

As will be detailed below, the invention also relates to the use of arecombinant nucleic acid molecule, of a vector, of a geneticallymodified host cell or of a non-human transgenic animal of the inventionor a non-human transgenic animal as generated by the method of theinvention or to the use of (a) neuronal determined cell(s) as isolatedby the method invention or of (an) isolated neuronal determined cell oran enriched or purified preparation of isolated neuronal determinedcells and progeny thereof as described herein for in vivo or in vitrotracking of newly generated neurons, of transplanted neuronal determinedcells or of migrating neuronal determined cells.

As described above, the present invention relates, inter alia, to thespecific uses of recombinant nucleic acid molecules encoding markergenes or reporter activities whereby the expression of said markers andreporters is driven by the regulatory sequence described in theinvention as DCX promoter. For example, a enzymatic activity may be usedas marker or reporter activity that can be detected in a host of thepresent invention, e.g. a host cell or a transgenic animal, as well asin tissues and organs of said animal. The systems provided in thisinvention allow also the in vivo and in vitro tracking of newlygenerated neuron. For example, in vivo imaging of newly generatingneurons during their “statu nascendi” is possible due to the provisionsof the present invention. Accordingly, an ideal in vivo system isprovided, which allows, inter alia, the test of environmentalconditions, psycho- and physiological conditions and lesions onneuron-generation and migration. Furthermore, substances/compounds andmixtures of compounds, such as potential therapeutics, may be tested fortheir potential to influence neurogenesis during development and in theadult by employing, inter alia, the transgenic animals of the invention.Since marker/reporter molecules are expressed in the host cells ortransgenic animals of the invention, their expression is specificallydetectable in proliferative neuronal determined/restricted cells andtheir expression is down-regulated in mature neurons. In vivo imaging ofthe marker/reporter or of the marker/reporter activity enables aqualitative and quantitative analysis of the neurogenic activity in thehost cell or the transgenic animal of the invention. In particular inthe transgenic animal of the invention, migration of neuronal precursorcells may be studied. However, it is also possible to employ cellsexpressing the recombinant nucleic acid molecule of the invention insuch studies. For example, such a host cell or isolated neuronaldetermined cell may be transplanted to a test animal and the fate of thetransplanted cell may be visualized or measured in vivo; Accordingly,the methods of the invention allow in vivo tracking of transplantedcells that carry the recombinant nucleic acid as described in theinvention, and their process of migration and neuronaldetermination/restriction/differentiation may be followed by means knownin the art. In addition to in vivo experiments, the invention can beused to follow migrating or neuronal determined/restricted cells in anin vitro/ex situ tissue preparation which comprises cells comprising arecombinant nucleic acid molecule of the invention. In this context, apreferred molecule to be expressed under the control of the regulatorysequence of the invention is luciferase or its derivatives.

The method of noninvasive optical imaging of, e.g. luciferase activityin living animals, such as transplanted or transgenic mice or rats, isavailable and known by skilled persons (Bhaumik and Gambhir, PNAS 99(2002), 377-382; Wu, Molecular Therapy 4 (2001), 297-306) and describedin Examples. Here, a cooled charged-coupled device (CCD) camera forcontinuous in vivo assessment is used. The CCD camera is mounted on alight-tight imaging chamber, that houses the host organism such astransgenic mouse expressing luciferase under the regulatory sequence ofthe invention. The advantages of using luciferase-based non-invasiveoptical imaging are several: 1. compared to calorimetric and fluorescentreporter proteins that require an external source of light forexcitation, bioluminescent luciferase gene(s), such as fireflyluciferase, does not need external light excitation, it self emits lightfrom yellow to green wavelengths in the presence of luciferin, ATP,magnesium and oxygen. Therefore, cells that activate expression of, e.g.luciferase under the regulatory sequence of the invention (neuronalrestricted/determined cells) are only luminescent for a transient periodof time and therefore allow multiple real-time measurements in one andthe same animal. 2. The fast rate of luciferase turnover (T ½=3 h) inthe presence of the substrate luciferin allows real time measurements.3. There is a linear relationship between luciferase concentration andthe emitted light in 7 to 8 orders of magnitude.

Therefore, the invention provides for in vivo and in vitro means andmethods which allow the screening of substances/candidate compoundscapable of influencing and/or modifying the fate and development ofneuronal cells, like neuronal precursor cells as well as neuronalrestricted/determined cells. Furthermore, with the means and methodsprovided herein, in particular the host cells and the transgenicnon-human animals, it is now possible to screen for substances/candidatecorresponds which promote neurogenesis. The substances are not onlyuseful in the prevention and therapy of neurological disorders anddiseases but are also useful in treating psychosises, learning or memorydisorders. Additionally, in vivo, in vitro and ex situ methods areprovided which allow the tracking of cells comprising an activatedregulatory sequence described in the invention. It is also envisagedthat cell migration assays be carried out, employing the recombinantnucleic acid molecule, the vectors, the host cells as well as cells ortissues derived from the transgenic animals of the invention. Theseassays may comprise grafting experiments. As a non-limiting example, itis envisaged that grafting experiments from one species to another arecarried out, e.g., the cells obtained from a transgenic mouse comprisingin its cells a recombinant nucleic acid molecule as described above (forexample the transgene luciferase, GFP. DsRed or the like) may be graftedinto, e.g., rat (brains) and migration behaviour, survival and/ordifferentiation of said grafted cells may be tested.

As mentioned above, in particular cell systems or transgenic animals (orcells, tissues or organs thereof) comprising the recombinant nucleicacid molecule described above may be employed in drug screenings, forexample in screenings for the detection and/or isolation of substancesand drugs which, either directly or indirectly activate the DCXregulatory sequence and which promote, inter alia,differentiation/determination programs in cells towards aneuron-phenotype or towards a neuron. As non-limiting example, atransgenic mouse model (or its cells, tissues or organs, like the brain)as described in the appended examples and expressing a marker gene (e.g.liciferase, DsRed, EGFP and the like) under the control of theregulatory sequence described herein may be used in drug screeningexperiments, in order to test the neurogenic potential (i.e. thepotential to generate new neurons in the adult brain) of a compound. Thedentate gyrus and the lateral ventricle/rostral migratorystream/olfactory bulb system are the two prominent regions of the adultbrain, which produce new neurons. Therefore, these two regions may beanalyzed in these mice for enhanced production of new neurons. Otherbrain regions, such as the neocortex, striatum, cerebellum, substantianigra and spinal cord, which have very low or undetectable adultneurogenesis, may be analyzed in order to test the ability of a compoundto induce de novo neurogenesis. In an exemplified experiment, theanimals receive a compound or a mixture of compounds to be tested eitherby, e.g., intracerebroventricular infusion via osmotic minipumps (Kuhn,J. Neurosci. 17 (1997), 5820-5829) or by peripheral administration (suchas intraperitoneal, subcutaneous or intravenous route). After severaldays of treatment the animals will be sacrificed and the brains areremoved. In order to measure the amount of marker (for example GFP)producing cells, several different detection methods may be used: Forexample, histological cell counting may be employed. Brains will besliced on a microtome and mounted on slides for histological analysis.With this method the number of marker (for example GFP) expressing cellsin the above mentioned brain areas may be counted either manually usinga fluorescent microscope or with assistance of automatic orsemiautomatic morphometric analysis systems. The goal is to generate thenumber of marker-expressing cells per brain structure. The data fromanimals, which received a drug, may be compared to control animals oranimals, which received a reference drug. Similarly, histologicalfluorescence detection may be employed. In order to avoid time-consumingmanual cell counting, a direct fluorescence intensity measurement may betaken to assess the expression level. Brains will be processed forhistological analysis and individual images from certain brain areas areanalyzed for marker (here fluorescence intensity using morphometricsoftware such as NIH Image. The goal is to generate a marker (e.g. GFP)intensity index for each mouse in order to compare animals, whichreceived a compound, to control animals or animals, which received areference drug. Biochemical methods may also be used: after tissuehomogenization, this procedure may allow the determination of totalmarker fluorescence per analyzed sample. Microdissected brain areas orwhole brain may be used to quantify the amount of green fluorescencemarker (e.g. GFP, EGFP and the like) from supernatant of tissuehomogenates. Supernatants may be analyzed in a fluorometer, such as afluorescent ELISA plate reader. Also, FACS-analysis may be employed. Asillustration, the brains of the transgenic animal expressing the markerunder control of the regulatory sequence of the invention aremicrodissected to analyze the above mentioned brain areas formarker-expressing cells. After tissue homogenization, single cellsuspension may be injected into a fluorescence-activated cell analyzerin order to determine the fraction of marker (e.g. GFP) expressing cellsamong the total population of cells in a given brain structure.

The non-human transgenic animals as well as the isolated cells andorgans from said animal are partially useful (like the host cellsdescribed herein) for the screening methods disclosed in this invention.They are partially useful in in vivo and in vitro assays for testing theneurogenic properties of candidate compounds.

Candidate agents for compounds capable of regulating neuronal determinedcell activity, neurogenesis and/or neuronal differentiation encompassnumerous chemical classes. It is, e.g. envisaged that antidepressants orgrowth factors be employed in the screening systems provided herein.Typical candidate agents are also typically organic compounds;preferably small organic compounds. Small organic compounds have amolecular weight of more than 50 Da yet less than about 2.500 Da,preferably less than about 1.000 Da, more preferably, less than about500 Da. Candidate agents comprise functional chemical groups necessaryfor structural interactions with lipids, proteins and/or DNA, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups, morepreferably at least three. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one ore more of the aforementionedfunctional groups. Candidate agents are also found among biomoleculesincluding peptides, proteins, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogues or combinationsthereof, and the like. Where the agent is or is encoded by a transfixednucleic acid, said nucleic acid is typically DNA or RNA. The candidatecompound itself may be an nucleic acid molecule, e.g. a DNA or an RNAencoding a potential candidate. Such candidates comprise for examplenucleic acid molecules encoding transcription factors orproteins/peptides involved in neuro-development and the like. Such acandidate may, however, also be a compound which inhibits the expressionof proteins. Such inhibiting candidates may, inter alia, be nucleic acidmolecules comprising, e.g., antisense oligonucleotides, RNAi and thelike. Candidate molecules which can be used in accordance with themethod of the present invention also include, inter alia, peptides,proteins, lipids, antibodies, aptamers, intramers or small organiccompounds.

As mentioned above, candidate agents may be obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds and biomolecules. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.In addition, known pharmacological agents may be subject to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc., to produce structural analogues.

The candidate molecule to be tested in the method of the presentinvention may be a single isolated substance as well as a plurality ofsubstances which may or may not be identical. Said candidatemolecules/compound(s) may be comprised in, for example, samples, e.g.,cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of influencing the activity of DCX regulatorysequences or not known to be capable of influencing the expression ofthe DCX regulatory sequences, respectively. The plurality of compoundsmay be, e.g., added to a sample in vitro, to the culture medium orinjected into the cell or a test animal, preferably a non-humantransgenic test animal.

If a sample (collection of candidate molecules) containing (a)compound(s) is identified in the method(s) of the invention, then it iseither possible to isolate the compound from the original sampleidentified as containing the compound in question or one can furthersubdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. It can then be determined whethersaid sample or compound displays the desired properties by methods knownin the art such as described herein. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances of similar chemical and/orphysical properties, and most preferably said substances are identical.The methods of the present invention can be easily performed anddesigned by the person skilled in the art, for example in accordancewith other cell based assays described in the prior art (see, e.g.,EP-A-0 403 506). Furthermore, the person skilled in the art will readilyrecognize which further compounds and/or cells may be used in order toperform the methods of the invention, for example,transfected/transduced host cells as described herein above. It is alsoenvisaged that the methods of the present invention are employed oncells, tissues or organs of the transgenic animal as described above.

The compounds/candidate molecules to be tested may also be functionalderivatives or analogues of known activators or inhibitors. Methods forthe preparation of chemical derivatives and analogues are well known tothose skilled in the art and are described in, for example, Beilstein,loc. cit. Furthermore, said derivatives and analogues can be tested fortheir effects according to methods known in the art and/or as describedherein. Furthermore, peptidomimetics and/or computer aided design ofappropriate activators or inhibitors of the expression the DCXregulatory sequence can be used, for example, according to the methodsdescribed herein. Appropriate computer systems for the computer aideddesign of, e.g., proteins and peptides are described in the prior art,for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak,Ann. N.Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),5987-5991. The results obtained from the above-described computeranalysis can be used in combination with the method of the inventionfor, e.g., optimizing known compounds, substances or molecules.Appropriate compounds/candidate molecules can also be identified by thesynthesis of peptidomimetic combinatorial libraries through successivechemical modification and testing the resulting compounds, e.g.,according to the methods described herein. Methods for the generationand use of peptidomimetic combinatorial libraries are described in theprior art, for example in Ostresh, Methods in Enzymology 267 (1996),220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.

In a further embodiment, the present invention provides for a method forthe preparation of a pharmaceutical composition for the treatment of aneurological disorder or disease comprising the steps of the method(s)described herein and, additionally, the formulation of (a) compound(s)capable of regulating neural stem cell activity, neurogenesis and/orneuronal differentiation as detected and/or obtained obtained by saidmethod(s) with a pharmaceutically acceptable carrier, excipients and/ordiluent. Examples of suitable pharmaceutical carriers, excipients and/ordiluents are mentioned herein above.

The methods described herein are particularly suited for automatedhigh-throughput drug screening using robotic liquid dispensingworkstations. Similar robotic automation is available forhigh-throughput cell plating and detection of various assay read-outs.

Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e. at zero concentration or below the limits ofassay detection.

These and other embodiments are disclosed and obvious to a skilledperson and embraced by the description and the Examples of the presentinvention. Additional literature regarding one of the above-mentionedmethods, means and uses, which can be applied within the meaning of thepresent invention can be obtained from the prior art, for instance inpublic libraries, e.g. with the use of electronic means. For thispurpose, public data bases, such as “Medline”, can be accessed via theinternet, for instance under the addresshttp://www.ncbi.nlm.nih.gov/PubMed/medline.html Additional data basesand addresses are known to a skilled person and can be taken from theinternet, for instance under the address http://www.lycos.com. Anoverview of sources and information regarding patents or patentapplications in biotechnology is given in Berks, TIBTECH 121 (1994),352-364.

The Figures show:

FIG. 1: Western blot analysis of antibodies directed againstdoublecortin (DCX)

-   -   Tissue homogenate from an adult rat olfactory bulb was separated        on a 12% polyacrylamid SDS-PAGE and transferred onto a        nitrocellulose membrane. Each lane, containing 2 μg of proteins,        was incubated into a different antibody solution. Lane 1 was        incubated in a solution containing goat anti-doublecortin C-18        antibody (Santa Cruz Laboratories), lane 2 was incubated in a        solution containing goat anti-doublecortin N-19 antibody (Santa        Cruz Laboratories) and the lane 3 was incubated in a solution        containing mouse anti-doublecortin antibody (Transduction        Laboratories). The immune complex was detected by        chemiluminescence. The goat anti-doublecortin C-18 antibody was        the more specific and recognized a single band of approximately        40 kDa, which corresponds to the expected molecular weight of        doublecortin.

FIG. 2: Time course of DCX expression in newly generated cells of theadult dentate gyrus

-   -   After injecting BrdU into 2 month-old rats, we determined in the        dentate gyrus granule cell layer over a period of 180 days the        changes in co-labeling of BrdU with DCX and NeuN. The time        course in (A) depicts the percentage of BrdU immuno-positive        cells co-labeling for DCX (diamonds), NeuN (squares) or DCX+NeuN        (circles). The data are presented as the percentages of        BrdU-positive per region, for each time point post BrdU        injection (n=4 animals per group, mean±S.E.M). (B-G)        Representative images from the dentate gyrus granule cell layer        depicting BrdU immunodetection (central block)(nuclear        immuno-reactivity), DCX (cytoplasmic immuno-reactivity detected        in the perikaryal regions and in the processes crossing the        granule cell layer (left block) and NeuN (right block) (nuclear        immuno-reactivity) at 2 hours (B), 4 days (C), 7 days (D), 10        days (E), 21 days (F), 60 days (G). Arrows indicate examples of        double labeling of BrdU with DCX and asterisks indicate double        labeling of BrdU with NeuN.

FIG. 3: DCX expression in proliferating cells

-   -   To confirm that DCX is expressed in proliferating cells, we used        co-labeling DCX with Ki-67, a marker for cells undergoing cell        division. (A) In the subgranular zone of the dentate gyrus (SVZ)        and (B) in the subventricular zone of the lateral ventricle wall        (DG) DCX/Ki-67 double labeling can be readily detected as        proliferative cell clusters. The Ki67 immuno-reactivity is        located in the nucleus, whereas the DCX immuno-reactivity is        cytoplasmic. Some of the Ki67 immuno-reactive nuclei are        indicated with arrows.

FIG. 4: Morphology of DCX expressing cells in the dentate gyrus

-   -   Two different phenotypes of DCX positive cells were found in the        dentate gyrus, which correlate with double labeling of BrdU at        certain time points after BrdU application: (A) Amorphous and        short branched cells in the subgranular zone with a tangential        orientation along the granule cells layer and (B) cells situated        at the base of the granule cell layer, with processes oriented        perpendicular reaching into the molecular layer. Arrows point to        cells co-labeled with BrdU and DCX.

FIG. 5: Doublecortin expression decreases in the aging dentate gyrus

-   -   Six time points ranging from 2 month-old animals to 21 month-old        animals were selected for analysis. These time points cover the        range over which the age dependent decrease in hippocampal        neurogenesis has been previously described (Kuhn, J, Neurosci,        16 (1996), 2027-2033). Images were obtained by confocal        microscopy using representative sections from the dentate gyrus.        Areas of greatest intensity of DCX were chosen from each section        for imaging. The DCX immunoreactivity can be appreciated in the        numerous processes crossing the granule cell layer. The NeuN        immunoreactivity is present in most of the granule cells nuclei,        thereby highlighting the granule cell layer.

FIG. 6: Morphology of DCX expressing cells during olfactory bulbneurogenesis

-   -   Similar to the dentate gyrus, multiple morphological phenotypes        of DCX immuno-reactive cells can be found in the subventricular        zone (RMS), rostral migratory stream (SVZ) and olfactory bulb        (OB), which correlate with double labeling of BrdU and DCX at        different time points after BrdU application: amorphous and        short branched cells in the subventricular zone undergoing cell        division, tangentially oriented cells in the rostral migratory        stream, representing migrating neuroblasts and cells in the        olfactory bulb extending neurites into the plexiform layer,        indicative of differentiating neuroblasts and young neurons. A        schematic anatomical drawing (center) depicts the anatomical        position from which the pictures were taken.

FIG. 7: Time course of DCX expression in newly generated cells of theadult olfactory bulb

-   -   After injecting BrdU into 2 month-old rats, we determined in the        olfactory bulb granule cell layer over a period of 180 days the        changes in co-labeling of BrdU with DCX and NeuN. The time        course in (A) depicts the percentage of BrdU immunopositive        cells co-labeling for DCX (diamonds), NeuN (squares) or DCX+NeuN        (circles). The data are presented as the percentages of        BrdU-positive per region for each time point post BrdU injection        (n=4 animals per group, mean±S.E.M.). (B-G) Representative        images of from the olfactory bulb granule cell layer depicting        BrdU (central block) (nuclear immuno-reactivity), DCX (left        block) (cytoplasmic immuno-reactivity detectable in the cell        soma and processes) and NeuN (right block) (nuclear        immuno-reactivity) at 2 hours (B), 4 days (C), 7 days (D), 10        days (E), 21 days (F), 60 days (G). Arrows indicate examples of        double labeling of BrdU with DCX. Note that no BrdU/DCX double        labeling can be found at 2 hrs and 4 days after labeling, since        the BrdU-positive neuronal precursor cells of the SVZ have to        migrate through the RMS and arrive in the olfactory bulb at        after approximately 4 days.

FIG. 8: Diagrams of the plasmids phuDCXpromoEGFP1 and phuDCXpromoDsRed2

-   -   The regulatory sequence of the DCX gene was subcloned in the        pEGFP-N1 and the pDsRed2-1 from BD Biosciences Clontech. The        cytomegalovirus (CMV) promoter was removed from the pEGFP-N1        vector prior to the insertion of the DCX regulatory sequences.        The regulatory sequence was subcloned in order to control the        expression of the fluorescent proteins EGFP (A) or DsRed2 (B).        Examples of the EGFP reporter gene under the control of the        human DCX regulatory sequence are the Seq ID NO:5 and 7.        Examples of the DsRed2 reporter gene under the control of the        human DCX regulatory sequence are the Seq ID NO:6 and 8.

FIG. 9: Alignment #1. Human vs Mouse regulatory sequences

-   -   Optimal global alignment between Sequence ID no:1 and Sequence        ID no:2 using the “fasta20u66, version 2.0u66” alignment        function available at Biology Workbench of the San Diego        Supercomputer Center.

FIG. 10: Diagram of the human vs mouse regulatory DCX sequences

-   -   A) The two sequences (SEQ ID NO 1: Human and SEQ ID NO 2: Mouse)        are illustrated with the exon sequences as black boxes. Only the        putative non-translated exon fragments are represented, i.e.        exon 1, exon 2, exon 3 and the beginning of exon 4. B) The        nucleic acid sequence outside the exons can be subdivided into        regions. The diagram illustrates the positions of these regions        and exons. The table gives the nucleotide positions of these        regions and exons in the mouse and human regulatory DCX        sequences, as well as the percentage of identity between the        corresponding regions and exons of the two sequences. The        fragment of the human DCX regulatory sequence comprised between        the nucleotide number 1166-3509 corresponds to the diagnostic        region.

FIG. 11: Alignment #2. Alignment of the diagnostic regions of the humanvs mouse DCX regulatory sequences

-   -   Optimal global alignment between Sequence ID no:3 (human) and        Sequence ID no:4 (mouse) using the “fasta20u66, version 2.0u66”        alignment function available at Biology Workbench of the San        Diego Supercomputer Center.

FIG. 12: Cloning strategy of the human DCX regulatory sequence

-   -   Schematic representation of the cloning of the human DCX        regulatory sequence using a PCR amplification approach (as        described in the Example II).

FIG. 13: Expression of the EGFP gene under the control of the human DCXregulatory sequence

-   -   A) Mouse embryonic day 14.5 dissociated forebrain cultures,        containing neuronal-restricted precursor cells, were plated over        poly-ornithin/laminin matrix for one week in the presence of NT3        and fetal calf serum. The culture were then transfected with the        phuDCXpromoEGFP1 vector. Two days after the end of the        transfection, the cultures were examined for the expression of        the EGFP reporter gene. In the right panel DAPI was used as a        nuclear counterstain. This photograph documents the presence of        cells with the morphology of young neuroblasts expressing the        reporter EGFP gene. Insert documents the various morphologies of        cells transfected with the reporter EGFP gene expressed under an        ubiquitous promoter (CMV; cytomegalovirus promoter).    -   B) Human fetal cortical stem cell cultures were treated and        transfected as in (A). The photograph documents the presence of        cells with the morphology of young neuroblasts expressing the        reporter EGFP gene.    -   C) Cultures of the COS7 cell line (ATCC number CRL-1651),        derived from SV40 transformed kidneys cells of Cercopithecus        aethiops and therefore not containing neuronal-restricted        precursor cells, were plated over poly-ornithin/laminin matrix        for 24 hours and transfected with the phuDCXpromoEGFP1 vector.        Two days after the end of the transfection, the cultures were        examined for the expression of the EGFP reporter gene. Few cells        expressed the EGFP reporter gene at the lower limit of        detection. In the right panel DAPI was used as a nuclear        counterstain. In contrast, identical culture transfected with        vector encoding the EGFP reporter gene under the control of an        ubiquitous promoter, i.e. the CMV promoter, strong expression of        the reporter gene could be detected in numerous cells (see C        inset).

FIG. 14: Strategy for the generation of transgenic mice expressing theEGFP gene under the control of the human DCX regulatory sequence

-   -   Schematic description of the generation of transgenic mice        expressing the EGFP reporter gene under the control of the human        DCX regulatory sequence (as described in the Example IV).

FIG. 15: Expression pattern of EGFP under the control of the human DCXregulatory sequence in two lines of transgenic mouse

-   -   Detection of the EGFP fluorescent signal in sagital brain        sections of 1 month-old huDCXpromoEGFP1 trangenic mice from the        line 299 and line 303. The EGFP reporter gene signal can be        detected in newly generated neuronal restricted precursors, in        particular in regions involved in active neurogenesis, i.e. in        the dentate gyrus and in the subventricular zone-olfactory bulb        axis. A-F) transgenic mouse from line 299. A-C) EGFP signal        detected in the olfactory bulb (A), cortex (B), dentate gyrus        (C), respectively. D-F) counterstain using the DNA binding        fluorochrome, DAPI, in the same visual fields as in A-C. G-L,        transgenic mouse from line 303. G-I) EGFP signal detected in the        olfactory bulb (G), cortex (H), dentate gyrus (I), respectively.        J-L) counterstain using the DNA binding fluorochrome, DAPI, in        the same visual fields as in G-I.

FIG. 16: Immunocharacterization of the huDCXpromoEGFP1 transgenic mouse

-   -   The olfactory bulb of a one month-old huDCXpromoEGFP1 transgenic        mouse from the line 303 was processed for immunohistochemistry        using antibodies directed against the doublecortin polypeptide        or the GFAP polypeptide. Photographs A to C document the        co-localization of the EGFP reporter gene expression, under the        control of the human DCX regulatory sequence, with the        endogenous expression of the mouse doublecortin. The arrows        point to some cell somata to allow for the comparison of the (B)        EGFP reporter signal with the (A) mouse endogenous doublecortin        expression. C) shows an overlay of the signals documented in        panels A and B. The EGFP reporter polypeptide is localized        preferentially in the cell soma, whereas the doublecortin        polypeptide distributes more significantly in the cell        processes. Photographs D to F document the absence of        co-localization of the EGFP reporter gene expression, under the        control of the human DCX regulatory sequence, with the        expression of GFAP, a polypeptide expressed in astrocytes. The        arrows point to (D) astrocytes detected by the anti-GFAP        antibody. These cells did not express the EGFP reporter gene as        revealed by the absence of EGFP fluorescent signal at the        corresponding coordinates (E). F) shows an overlay of the        signals documented in panels D and E.

FIG. 17: Putative binding sites for transcription factors on SEQ ID NO.:1 and SEQ ID NO.: 2

-   -   Putative binding sites for transcription factors on SEQ ID NO:1        and SEQ ID NO:2 as determined by computer analysis are shown (as        described in the text).

FIG. 18: Specific activity of the human DCX regulatory sequence in cellsof neuronal lineage

-   -   Different cell types (HEK293, COS7, CTX TNA2, N20.1, D283Med,        Neuro2A and MEF E10,5) were transiently transfected with        pEGFP-N1 or phuDCXpromoEGFP1 and analyzed for expression of        EGFP.

Whereas all the cell types can be transfected and express EGFP undercontrol of the ubiquitous CMV promoter (left block), the DCX regulatorysequence drives reporter gene expression specifically in cells ofneuronal lineage (right block).

FIG. 19: Quantitative analysis of promoter activity

-   -   Different cell types (HEK293, COS7, CTX TNA2, N20.1, D283Med,        Neuro2A and MEF E10,5) were transiently transfected with        pEGFP-N1 or phuDCXpromoEGFP1 and analyzed for expression of        EGFP. A) The percentage of cells that express EGFP is analyzed        and shown. B) The expression efficacy (calculated as the        percentage of cells that express EGFP after transfection with        phuDCXpromoEGFP1 relative to the percentage of cells that        express EGFP after transfection with pEGFP-N1) is shown. Note        the high level of expression efficacy in cells of neuronal        lineage.

FIG. 20: Cell type analysis of cells present in MEF E10,5 cells

-   -   MEF E10,5 cells are analyzed for marker expression after one        week of differentiation. A) shows immunfluorescence staining of        different markers present in the culture. B) shows a        quantitative analysis of the percentages of cells expressing the        various markers in MEF E10,5 cell cultures.

FIG. 21: Frequency of DCX co-expression with various markers in MEFE10,5 cells after 1 week of differentiation

-   -   Note that DCX expression colocalizes with neuronal determined        cell types, and not glia (GFAP, GaIC) or not stem cells        (Nestin).

FIG. 22: Cell type analysis of cells in which the human DCX regulatorysequence is active

-   -   MEF E10,5 were transiently transfected with phuDCXpromoEGFP1 and        immunostained for different markers. EGFP positive cells        colabeled with endogenous markers for neuronal determined cells,        but not with stem cells or glial markers.

FIG. 23: Quantitative analysis of experiments In FIG. 22

-   -   The frequency of presence of the various markers shown in FIG.        22 was quantified in MEF 10,5 cell expressing EGFP following        transfection with phuDCXpromoEGFP1. Additionally, for        comparative purposes, the frequency of DCX-EGFP expression in        MEF 10,5 cells after pEGFP-N1 transfection is shown.

FIG. 24: Deletion constructs of SEQ ID NO.: 1

-   -   The deletion fragments presented in this figure were used to        control the expression of the EGFP reporter gene as shown in the        Seq ID NO: 20 to 25.

FIG. 25: Specific activity of the deletions constructs of FIG. 24 whentransfected in MEF E10,5 cells

-   -   MEF E10,5 were transiently transfected with different deletion        constructs and the percentage of EGFP positive cells that        co-label with DCX was analyzed.

FIG. 26: Ciliary body and RPE derived cells express progenitor markersin vitro

-   -   (A) RT-PCR for β III Tubulin, nestin, Pax6, Dcx and musashi with        GAPDH as standard. Products are 140 bp for each primer pair.        (B)-(D) Immunostaining for the neural stem cell marker nestin        with nuclear counterstain DAPI on RPE derived cells after 21 DIV        under differentiation conditions (NB/B27+5% FCS).

FIG. 27: Neuronal phenotype and morphology in RPE derived cells andquantification of differentiation in CB and RPE cells

-   -   Cell derived from RPE cultures of passage number 3 grown for 7        days on laminin coated glass cover slips are shown. (A)-(C)        Immunostaining for β III Tubulin with nuclear counterstain DAPI.        The morphology of the cell is of neuronal character. (D)-(F)        Double immunostaining for β III Tubulin and the neuronal        precursor marker Dcx with nuclear counterstain DAPI. (G)-(I)        Immunostaining for β III Tubulin (H) and Dcx (G) with nuclear        counterstain DAPI in RPE cells grown under proliferation        conditions on collagen coated plastic. The cell displays a more        epithelial morphology and does not coexpress Dcx. (J), (K)        Quantification of the percentage of cells expressing Dcx and β        III Tubulin in CB and RPE cultures after 7 DIV under        differentiation conditions. Data is expressed as mean value        +/−standard error of the mean (S.E.M.).

FIG. 28: Enrichment of cells expressing DsRed2 derived from an animal asdescribed in Example IV by FACS-sorting

-   -   Cell cultures originating from dissociated brains of neonatal        huDCXpromoDsRed2 transgenic mice analyzed immediately after        FACS-sorting. (A) Phase contrast photograph of a culture        composed of cells FACS-sorted for the presence of DsRed2        fluorescence. (B) Fluorescence signal obtained from the DsRed2        reporter protein detected in the same observation field as in A.        Some cells expressing the DsRed2 reporter gene are marked with        arrows, whereas some cells devoid of DsRed2 are marked with an        arrow head. Note that more the 99% of the cells are expressing        the DsRed2 reporter gene. (C) Phase contrast photograph of a        culture composed of cells FACS-sorted for the absence of DsRed2        fluorescence. (D) Fluorescence signal obtained from the DsRed2        reporter protein detected in the same observation field as in C.        Note that more the 99% of the cells are negative, i.e. do not        express the DsRed2 reporter gene. Scale bar in D represents 200        μm.

FIG. 29: Cells expressing a fluorescent gene (EGFP or DsRed2) derivedfrom an animal as described in Example IV enriched by FACS yieldsDoublecortin-positive cells

-   -   Cell cultures originating from dissociated neonatal brains of        huDCXpromoDsRed2 or nestin-EGFP transgenic mice. Cells were        FACS-sorted into positive and negative populations in respect to        their expression of the reporter genes DsRed2 and EGFP        respectively. The sorted populations were maintained in culture        for 1 day before fixation as described in EXAMPLE VI. Panels A        to C document that cells derived from the huDCXpromoDsRed2        transgenic mice sorted for the presence the DsRed2 reporter (A)        resulted in a culture with >90% of cells expressing doublecortin        (B), nuclear counterstaining with Dapi is shown in (C). Panels D        to F document that cells derived from the huDCXpromoDsRed2        transgenic mice sorted for the absence the DsRed2 reporter (D)        resulted in a culture with <5% of cells expressing doublecortin        (B), nuclear counterstaining with Dapi is shown in (F). Panels G        to I document that cells derived from the nestin-EGFP transgenic        mice sorted for the presence the EGFP reporter (G) resulted in a        mixed culture with some of cells expressing doublecortin (H),        nuclear counterstaining with Dapi is shown in (I). Panels J to L        document that cells derived from the nestin-EGFP transgenic mice        sorted for the absence the EGFP reporter (J) also resulted in a        mixed culture with some of the cells expressing doublecortin        (K), nuclear counterstaining with Dapi is shown in (L). The        level of expression of the EGFP reporter gene in the nestin-EGFP        FACS-sorted cells is significantly downregulated after 1 day of        culture on coverslip. The presence of cellular debris is        responsible for some unspecific binding of the doublecortin        antibody. Only cells with a normal nuclear morphology were        considered. Scale bar in (L) represents 100 μm.

FIG. 30: huDCXpromoEGFP transgenic mice to study variations inneurogenesis levels

-   -   EGFP fluorescence in the dentate gyrus of different        huDCXpromoEGFP1 transgenic mice is shown. A) shows a section        from a 1 month-old mouse, B) shows a section of a 2 month-old        mouse, C) shows a section of a 12 month-old mouse. Note the        decline of EGFP fluorescence intensity as a function of age        according to the reported age-related decrease of neurogenesis.        D and E) show sections of three month-old mice D) a control        mouse, and E) a mouse 7 days after experimentally-induced        epileptic seizures. Note the seizure-related up-regulation of        EGFP signal in E) according to the reported increase of        neurogenesis occurring after seizure activity. F) shows the        results of a quantitative analysis.

The examples illustrate the invention.

EXAMPLE I Transient expression of doublecortin (DCX) during adultneurogenesis

a) Methods and Materials Used in this Study:

Animals and BrdU Injections

Female Wistar rats (Charles River-Wiga, Sulzfeld, Germany) were kept innormal light dark cycle (12 hour light/12 hour dark) and had free accessto food and water. All animal experiments were approved by theuniversity's animal care commission and by local government and wereconform with NIH guidelines and German law. Animals received at 2 monthof age intraperitoneal injections with BrdU (50 mg/kg body weight).Animals perfused at 2 hours, 4 and 7 days after BrdU treatment receivedonly a single BrdU injection. The animals that were sacrificed at 10,14, and 21 days, 1, 2, 3, 4, 6, 9, 14, and 19 months after BrdUinjection received daily injections on 4 consecutive days. Each timepoint consisted of 3 animals.

SDS-PAGE/Western Blot

The olfactory bulb from a 2 month-old female Wistar rat(Charles-River-Wiga, Sulzfeld, Germany) was homogenized in SUB buffer(0.5% SDS, 8 M urea, 2% β-mercaptoethanol) and centrifuge at roomtemperature for 10 minutes at 12 000×g to remove the insoluble debris.The protein concentration in the supernatant was determined usingBradford reagent (Sigma, St. Louis, Mo., USA). The sample (2 μg/lane)was electrophoresed in a 12% polyacrylamide SDS-PAGE and transferredonto a nitrocellulose membrane (Schleicher and Schuell, Dassel,Germany).

Membranes were placed into blocking buffer (20 mM Tris-HCl, pH 7.3, 0.9%NaCl, 1% Teleostean gelatin (Sigma, St. Louis, Mo., USA), and 0.1%Tween-20) for 1 hour at room temperature. The same buffer served forantibody dilutions, as well as for washes. For detection of DCX, thefollowing primary antibodies were used: goat anti-DCX C-18 (1:500, SantaCruz Labs, Santa Cruz, USA), goat anti-DCX N-19 (1:500, Santa Cruz Labs,Santa Cruz, USA), and mouse anti-DCX (1:500, Transduction Labs,Lexington, USA). The blots were incubated in primary antibody solutionsovernight at 4° C. on a shaking table. The following day, the blots werewashed and further incubated with peroxidase-conjugated species-specificsecondary antibody for 2 hours at room temperature (rabbit anti-goat1:5000 (Sigma, Taufkirchen, Germany) and donkey anti-mouse 1:5000(Jackson ImmunoResearch, West Grove, USA). Blots Were washed and theimmune complex was detected using the SuperSignal West Picochemiluminescent substrate (Perbio, Bonn, Germany) according to themanufacturer's protocol.

Histology

The animals were deeply anesthetized and perfused transcardially with 4%paraformaldehyde in 100 mM phosphate buffer, pH 7.4. The brains weredissected, immersed overnight in fixative, and transferred to 30%sucrose/100 mM phosphate buffer, pH 7.4 for at least 48 hours. Brainswere cut into 40 μm sagittal sections using a sliding microtome.Sections were stored at −20° C. in cryoprotectant solution untilstaining (25% v/v glycerol, 25% v/v ethylene glycol, and 0.05M phosphatebuffer, pH 7.4).

Immunofluorescence

To allow for a better penetration of antibodies in areas of highneuronal density, free-floating sections were placed in 1% TritonX-100/TBS (Tris-buffered saline: 0.1M Tris-HCl pH 7.4/0.9% NaCl)solution for 15 minutes followed by three consecutive 5 minute washeswith TBS. For detection of incorporated BrdU, the sections weresubjected to the following procedure: incubation in 0.3M NaCl/30 mMCitrate Buffer pH 7.0/50% (v/v) formamide at 65° C. for 2 hours, rinsein 0.3M NaCl/30 mM Citrate Buffer pH 7.0, incubation in 2N HCl at 37° C.for 30 minutes, rinse in 0.1M borate buffer (pH 8.5) for 10 minutes,rinse in TBS. Sections were blocked in TBS/3% donkey serum/0.1% Triton-X100 (TBS-DS-TX) for 30 min, followed incubation with primary antibodiesin TBS-DS-TX for 48 hours at 4° C. The following primary antibodydilutions were used: rat anti-BrdU (1:500 Accurate, Westbury, USA),mouse anti-NeuN (1:500, Chemicon, Temecula, USA), goat anti-DCX C-18(1:500, Santa Cruz Labs, Santa Cruz, USA), rabbit anti-KI67 (1:500,Novacastra Laboratories Ltd., Newcastle Upon Tyne, UK). The sectionswere then rinsed in TBS three times for 10 minutes, and then incubatedwith secondary antibodies in TBS-DS-TX for 2 hours. The followingfluorochrome-conjugated secondary antibodies were used: donkeyanti-rat-CY5 F(ab)₂ fragment, donkey anti-mouse-rhodamineX F(ab)₂fragment, donkey anti-goat-FITC F(ab)₂ fragment and donkeyanti-rabbit-FITC (all 2 μg/ml, Jackson ImmunoResearch, West Grove, USA).After several washes in TBS, sections were mounted on gelatin-coatedglass slides and coverslipped using Prolong (Molecular Probes, Eugene,Oreg.).

Quantification

Analysis was performed using a confocal microscope (TCS-NT, LeicaMicrosystems, Bensheim, Germany) equipped with a 40×PL APO oil objective(1.25 NA) and a pinhole setting that corresponded to a thickness of thefocal plane of less than 2 μm. Randomly selected BrdU-positive cellswere analyzed in their entire z-axis in order to exclude falsedouble-labeling due to an overlay of signals from different cells (Kuhn(1997), loc. cit.). A minimum of 50 BrdU-positive cells per region ofinterest were examined for co-labeling with DCX and NeuN in each animaland time point. Data are presented as the average percentage ofBrdU-positive cells, which co-labeled for DCX, NeuN or DCX/NeuN(Mean+/−S.E.M.).

b) Selection of Doublecortin Antibody by Western Blot Analysis

The specificity of three commercially available antibodies directedagainst DCX was examined by western Blot analysis. A band of 40 kDa,consistent with the molecular weight of the DCX protein, was detected byall three antibodies (FIG. 1). Nevertheless, the antibody directedagainst the C-terminus of DCX (Santa Cruz Labs, Santa Cruz, USA) provedto be the most specific (FIG. 1, Lane 1). The two other antibodies (FIG.1 lane 2 and 3) detected several other proteins at higher molecularweights. One such band could be doublecortin-like kinase (DCLK), arelated protein sharing 85% homology with the N-terminus of DCX (Ohara,DNA Res. 4 (1997), 53-59). DCLK is a microtubule-associated proteinkinase also expressed in brain. Because of its higher specificity, thegoat anti-DCX C-18 antibody was chosen for immunohistological analysisof the tissue sections. Accordingly, the present study documents thatonly highly specific antibodies directed against DCX can be employed inorder to elucidate temporal and spacial expression of said DCX.

c) Doublecortin Expression During Adult Hippocampal Neurogenesis

Time Course Analysis

The pattern of DCX expression within newly generated cells was examinedby immunofluorescence labeling of DCX and BrdU in the neurogenic regionsof the adult rat brain different time points after BrdU administration.We first quantified the number of BrdU-positive cells that expressed DCXwithin the dentate gyrus (FIG. 2). At the earliest time point analyzed,i.e. 2 hours post-BrdU administration, we observed co-labeling of DCXwithin 60% of the newborn cells (FIG. 2). The fact that the majority ofBrdU-positive cells were expressing DCX at the earliest time pointstrongly suggested that proliferating cells were already expressing DCX.To further substantiate this observation, frequent co-labeling of DCXwith Ki67 could be demonstrated. Ki67 is an antigen enriched inproliferating cells during DNA synthesis and mitosis, in the hippocampusand lateral ventricle wall (FIG. 3). Between the seventh and tenth daypost-labeling the percentage of BrdU-positive cells expressing DCXfurther increased to more than 90% (FIG. 2). Thereafter, DCX expressionwas rapidly downregulated. It was observed in only 2% of theBrdU-positive cells by one month and became undetectable by two monthsafter labeling.

Towards the final stage of neuronal differentiation, the newborn cellsbegin to express proteins typically present in mature neurons such asthe nuclear neuronal marker NeuN, neuronal-specific enolase (NSE), orcalbindin (Cameron (1993), loc. cit.; Kuhn (1996), loc.cit and Kuhn, J.Neurosci, 17 (1997), 5820-5829). Once induced, these markers areexpressed throughout the lifetime of the neuron. For that reason, weused NeuN to determine when newly generated neuronal precursors becomemature and to what extent NeuN and DCX expressions overlap. BrdU-labeledcells immunoreactive for NeuN were first detected in the hippocampus at10 days after BrdU injection (FIG. 2). The majority of the NeuN positivecells co-expressed DCX between Day 10 and 14, thereafter NeuN/DCXco-labeling was not detectable anymore. The percentage of BrdU-positivecells expressing NeuN increased to about 80% one month after labelingand increased further to more than 90% at later time points analyzed(FIG. 2). Adjacent non-neurogenic regions, the hilus and molecularlayers of the dentate gyrus, were analyzed as control areas. Theco-localization of BrdU-positive cell bodies with DCX or NeuN was notdetected within these regions.

Morphology of DCX Expressing Cells

It is interesting to note that the morphology of DCX-expressing cellschanged as neuroblasts matured (FIG. 4). Two cellular morphologies wereobserved. Within the first days after BrdU-labeling, DCX-positive cellsformed clusters in the subgranular zone adjacent to the inner margin ofthe granule cell layer. Some of these cells were without definedprocesses, whereas others resembled neuroblasts with processes orientedparallel to granule cell layer (FIG. 4 a). Later, at about 10 days afterBrdU labeling, DCX-positive cells were integrated into the granule celllayer and displayed processes spanning the entire layer and further intothe molecular layer (FIG. 4 b).

d) Doublecortin Expression in the Aging Dentate Gyrus

An age-dependent decrease of neurogenesis within the granule cell layerof the dentate gyrus has previously been reported (Kuhn (1996), loc.cit.). Consequently, we performed an analysis of DCX expression as afunction of age. The highest incidence of DCX immunoreactivity wasobserved in the younger animals examined, i.e. 2 month-old rats. Thetotal amount of cells expressing DCX notably decreased by 11 months ofage, and very few DCX positive cells were detectable in 21 month-oldrats (FIG. 5). The reduction of DCX-expressing cells in the dentategyrus is consistent with and substantiates the reported age-dependentneurogenesis decrease.

The current study analyzed the time course of DCX expression in adultborn cells of the dentate gyrus and the lateral ventricle wall/olfactorybulb. DCX is to a high degree expressed in dividing cells (FIG. 2 andFIG. 3). DCX is not detected in the embryonic ventricular zone duringcortex development (Gleeson, Neuron 23 (1999), 257-271), but it isexpressed in proliferating cells of the adult SVZ and hippocampus.Apparently, fetal and adult neural progenitors cells have differentmolecular identities and properties.

e) Doublecortin Expression in the Granule Cell Layer of the OlfactoryBulb

Newly generated neuroblasts of the olfactory bulb arise in thesubventricular zone (SVZ) of the lateral ventricle wall, whereproliferation of neural stem cells and neuronally committed progenitorstakes place (Doetsch (1997), loc.cit.). The new cells form chains ofmigrating cells, which converge to form the rostral migratory stream.After reaching the olfactory bulb (OB) the cells integrate into thegranule cell layer and periglomerular region and begin to express matureneuronal markers.

Subventricular Zone/Rostral Migratory Stream

DCX is already strongly expressed in the subventricular zone (FIG. 6).Similar to the hippocampus, numerous cells in the subventricular zonewere double-labeled for DCX and the proliferation marker Ki-67,indicating frequent cell division of neuroblasts (FIG. 3). Themorphology of the DCX-expressing cells in the subventricular zone ismostly bipolar with short processes (FIG. 6). The DCX-positive cells areorganized in chain-like structures, which has been previously beendescribed for migratory neuroblasts (Lois, Science 271 (1996), 978-981).In the rostral migratory stream, the DCX-expressing neuroblasts have anelongated morphology with a leading process, consistent with neuroblastmigration towards the OB (FIG. 6).

Olfactory Bulb

Finally, in the OB the DCX-expressing cells adopt a more complexmorphology, similarly to the one observed for the cell integrating intothe cell granular layer of the hippocampus (FIG. 6). Consequently,BrdU-labeled cells coming from the SVZ were first detected in theolfactory bulb four days after BrdU injection. This interval reflectsthe time required for neuroblasts to migrate from the SVZ, where theyarise and incorporate BrdU, to the OB. At four days after labeling, only2% of the BrdU-labeled cells in the OB were found to express DCX. Atthis time point, the percentage is relatively low since most to theBrdU-labeled cells observed in the OB result from in situ cell divisionof non-neuronal cells. Over the next days, as the bulk of newlygenerated neuroblasts from the SVZ reach the OB, this percentageincreased rapidly. Ten days after labeling, approximately 70% of theBrdU-positive cells in the OB were expressing DCX (FIG. 7). Similar tothe hippocampus, the number of BrdU-positive cells expressing DCX withinthe OB decreased to very low levels by 1 month after labeling andremained undetectable throughout the subsequent time points. Theco-localization of BrdU with NeuN was first detected at 14 days afterBrdU injection. At this time, 24% of the BrdU-labeled cells expressedNeuN (FIG. 7). The percentage of BrdU-labeled cells expressing NeuNincreased to nearly 90% by 1 month and remained at this level in thelater time points analyzed.

f) Doublecortin is a Useful Marker for Neuronal Determined Cells and theDetection of Neurogenesis, Independent from Pre-Labeling Methods

Within the first 10 day after BrdU-labeling, the percentage of DCXpositive cells among the new cells increased to about 90%. However, oncethe cells become older and begin to express markers for mature neuronsDCX is downregulated to undetectable levels. Although it is not possibleto predict the fate of individual DCX positive cells from histologicalanalysis, it is very intriguing that the percentage of BrdU/DCXco-labeling is almost identical to the percentage of new cells whichlater differentiate into neurons (>90% BrdU/NeuN co-labeling, see FIGS.2 and 7). This shows that DCX is transiently expressed in neuronallycommitted cells and therefore can serve as an indicator for adultneurogenesis.

DCX immunoreactivity is not exclusively found in immature neurons, buthas been reported to be occasionally present in neurons withdifferentiated morphology within non-neurogenic regions, includingcortex, striatum and corpus callosum (Nacher (2001), loc. cit.).However, in the current study only very low levels of DCX expressionoutside the neurogenic regions was observed. One reason for thisdiscrepancy might be the use of different antisera against DCX betweenstudies. As shown by comparative western blot analysis, antibodiesrecognize to a different degree additional antigens and may thereforenot reflect DCX alone (FIG. 1). Another explanation for occasional DCXexpression outside the neurogenic regions could be that neurogenesis mayperhaps exist in these regions.

This hypothesis is supported by recent evidences that induction ofneurogenesis in the cortex and striatum under pathological conditions isassociated with the migration of DCX-positive neuronal progenitors fromthe lateral ventricle wall. (Magavi, Naute 405 (2000), 951-955;Arvidsson, Nat. Med. 8 (2002), 963-970). Moreover, it has been suggestedby other studies that adult neurogenesis is a common feature of severalbrain regions, including the cortex and amygdala (Gould, PNAS 96 (1999),5263-5267; Pencea, Exp. Neurol. 172 (2001), 1-16; Bernier PNAS 99(2002), 11464-11469) although at very low levels.

Since DCX is a protein, which binds, bundles and stabilizesmicrotubules, it could play a role into various neuronal cytoskeletondepending scenarios including migration of neuronal precursor cells,nuclear translocation, and axonal and dendritic maturation. Here, it wassurprisingly found and documented that, due to the time course of DCXexpression during neuronal maturation in the adult brain, DCX is presentnot only in proliferating and migrating but also differentiatingneuronal progenitors, e.g. during dendritic elongation and arborization(FIGS. 2, 4 and 6). LIS1, a protein functionally similar to DCX andassociated with neuronal migration, has been shown to regulateneuroblast proliferation, nuclear translocation and positioning as wellas dendritic elaboration and axonal transport in drosophila (Swan, Nat.Cell Biol 1 (1999), 444449; Lei, Dev. Biol. 226 (2000), 57-72; Liu, NatCell Biol. 2 (2000), 776-783 as well as Leveter Trends Neurosci 24(2001), 489-492) indicating that microtubule-associated proteins playimportant roles in most cellular function involving cytoskeletalrearrangement.

In addition to characterizing the role of DCX in the adult brain,results documented here improve neurogenesis-tests and -analysis on amethodological level. Traditional techniques to study neurogenesis haveutilized labeling with tritiated thymidine, BrdU or retrovirus, inconjunction with various neuronal markers to address the nature and fateof neural stem cell population. The limitations of these protocols, inparticular the need to perform in vivo pre-labeling, have made theidentification of a specific and early marker for neuronal lineage ofgreat importance. In this study, it is shown that DCX is detected inneuroblasts within the first month after their emergence. The temporalexpression pattern and specificity of DCX within the adult neurogenicregions suggest that DCX is a useful marker for the detection ofneurogenesis independent from pre-labeling methods, like BrdUincorporation. Such a neurogenesis marker provides a valuable toolespecially under circumstances that prohibit the use of BrdU, forinstance in postmortem analysis of human brain tissue.

EXAMPLE II Cloning of the Human DCX Regulatory Sequence

PCR Amplification of the Genomic Fragment Comprising the Human DCXRegulatory Sequence

Using the partial human X chromosome sequence available under theGenBank accession number AL450490, two oligonucleotides (oligo no:1:AACACC TAT TAA TGC CCA; SEQ ID NO.: 9 and oligo no:2:TCA GAG ACC TGA GCGTGG GAG AA; SEQ ID NO.: 10) were designed for the PCR amplification ofan approximately 3.5 kilobase pairs fragment. The sequence of theamplified fragment was chosen in order to comprise approximately 1.5kilobase pairs of genomic sequence upstream the DCX putative exon 1, thegenomic sequence corresponding to the DCX first three putative exons,the genomic sequence corresponding to the three first DCX introns and afew base pairs upstream from the start position of DCX protein codingsequence located in the putative exon 4.

The PCR amplification on human genomic DNA was performed using ExpandHigh Fidelity PCR System kit (Roche) according to the manufacturer'sprotocol. The following PCR protocol was used: 94° C. 3 minutes 1 cycle94° C. 45 seconds 30 cycles 59° C. 30 seconds 72° C. 3 minutes 72° C. 8minutes 1 cyclePreparation of the PCR Product Comprising the Human DCX RegulatorySequence for Subcloning.

A single PCR product of approximately 3.5 kilobase pairs was obtained asvisualized on a 1% agarose gel electrophoresis. The DNA present in theband of 3.5 kilobase pairs was recovered using the MiniElute GelExtraction kit (Qiagen). The purified fragment was blunt-ended with theDNA polymerase I Klenow fragment without the addition of free dNTPs. Theblunt-ended fragment was then phosphorylated using the T4 polynucleotidekinase in the presence of ATP. This processed DNA fragment correspondsto the Sequence ID no:1 and was further used for subcloning.

Preparation of the Cloning Vector Containing the Reporter Gene EGFP

The CMV promoter from the plasmid pEGFP-N1 (Clontech) was deleted usingthe restriction enzymes AseI and NheI. A DNA fragment of approximately4.1 kilobase pairs corresponding to the linearized pEGFP-N1 vectorwithout the CMV promoter was isolated from a 1% agarose gel followingelectrophoresis using the MiniElute Gel Extraction kit (Qiagen). Theisolated DNA fragment was blunt-ended using the DNA polymerase I Klenowfragment in the presence of 33 microM of free dNTPs. The resulting DNAfragment was further re-circularized using the T4 DNA ligase resultingin a promoterless pEGFP-N1 vector of approximately 4.1 kilobase pairs.

Subcloning of the PCR Fragment Comprising the Human DCX RegulatorySequence into the Promoterless pEGFP-N1 Vector

For the insertion of the human DCX regulatory sequence, the promoterlesspEGFP-N1 vector was linearized using with the restriction enzyme Smal.The DNA fragment was dephosphorylated using calf intestinal alkalinephosphatase. The linearized fragment was isolated by electrophoresis ina 1% agarose gel followed by extraction using the MiniElute GelExtraction kit (Qiagen). The resulting linear vector and the DNAfragment corresponding to the human DCX regulatory sequence were ligatedtogether into a circular plasmid using the T4 DNA ligase according tothe manufacturer's protocol (New England Biolabs). In this construct ofapproximately 7.7 kilobase pairs, phuDCXpromoEGFP1 Sequence ID no:7, thehuman DCX regulatory sequence controls the expression of the EGFP gene(FIG. 8A).

Subcloning of the PCR Fragment Comprising the Human DCX RegulatorySequence into the pDsRed2-1 Vector

The pDsRed2-1 vector (Clontech) was digested with the restrictionenzymes Sall and BamHI. A DNA fragment of approximately 4.1 kilobasepairs, was purified by electrophoresis in an 1% agarose gel followed byan extraction using the MiniElute Gel Extraction kit (Qiagen). The DNAfragment was dephosphorylated using calf intestinal alkalinephosphatase. The phuDCXpromoEGFP1 vector was digested with therestriction enzymes SalI and BamHI. A DNA fragment of approximately 3.5kilobase pairs, encoding the human DCX regulatory sequence, was purifiedby electrophoresis in an 1% agarose gel followed by an extraction usingthe MiniElute Gel Extraction kit (Qiagen). The purified fragment wasligated to the prepared pDsRed2-1 vector using the T4 DNA ligaseaccording to the manufacturer's protocol (New England Biolabs). In theresulting vector, phuDCXpromoDsRed2 Sequence ID no:8, of approximately7.6 kilobase pairs, the human DCX regulatory sequence controls theexpression of the DsRed2 reporter gene. The cloning protocol isschematized in FIG. 12.

EXAMPLE III Expression of the phuDCXpromoEGFP1 Vector in Cell Cultures

Mouse Embryonic Forebrain Cell Cultures

Pregnant C57BL/6Ncrl mouse females (Charles River Laboratories) weresacrificed by cervical dislocation. The uterus were removed and immersedin ice cooled Dulbecco's phosphate buffered saline solution (DBPS).Embryonic day 12.5 to 14.5 embryos were released from the uterus, theforebrain was dissected and separated from surrounding tissues. Thedissected forebrain was washed once in ice-cooled DPBS, transferred to apetri dish and dissociated mechanically with a scalpel blade. Theresulting preparation was washed in DPBS. Following 5 minutescentrifugation at 120×g, the pellet was resuspended in PPD-solutioncontaining 0.01% Papain (Worthington Biochemicals, England), 0.1%dispase II (Boehringer, Germany), 0.01% DNaseI (WorthingtonBiochemicals, England) and 12.4 mM MgSO₄ in HBSS without Mg⁺⁺/Ca⁺⁺ (PAA,Germany) and incubated for 30 to 40 minutes at 37° C. The cellpreparation was triturated every 10 minutes through a pipette.Dissociated cells were collected by 5 minutes centrifugation at 120×g.The pellet was resuspended in Neurobasal medium (Gibco BRL, Germany) andwashed three times. Finally, the cell preparation was resuspended inNeurobasal medium supplemented with B27 (Gibco BRL, Germany), 2 mML-Glutamine (PAN, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin(PAN, Germany), 2 μg/mL heparin (Sigma, Germany), 20 ng/mL bFGF-2 (R&DSystems, Germany) and 20 ng/mL EGF (R&D Systems, Germany) and maintainedat 37° C. in a 5% CO₂ containing humidified atmosphere. Growingsuspension cultures formed cell aggregates referred to as neurospheres.In order to dissociate these neurospheres for passaging, cultures werecentrifuged for 5 minutes at 120×g and the pellet was resuspended inAccutase (Innovative Cell Technologies, Inc.). The suspension wasincubated at 37° C. for 10 minutes. The dissociated aggregates wererecovered by centrifugation for 5 minutes at 120× and the cells reseededin the growing media described above.

Human Fetal Cortical Cell Cultures

These cultures originate from an 8 weeks post conception human brain.Description of the culture conditions have been previously described inSvendsen, Journal of Neuroscience Methods 85 (1998), 141-152.

Preparation of the Forebrain Cell Cultures for Transfection

Following dissociation of the cell aggregates with Accutase (InnovativeCell Technologies, Inc.) at 37° C. for 10 minutes, cells were seeded oncoverslips coated with poly-ornithin and laminin at a density of 7.5×10⁴cells per cm², in Neurobasal medium supplemented with B27 (Gibco BRL,Germany), 2 mM L-Glutamine (PAN, Germany), 100 U/mL penicillin, 0.1mg/mL streptomycin (PAN, Germany), 10 ng/mL NT3 and 1% fetal calf serumand maintained for 1 week at 37° C. in a 5% CO₂ humidified atmosphere.

COS7 Cell Cultures

COS7 cell were obtained from ATCC U.S.A. (number CRL-1651) andmaintained in culture in Dulbecco's modified Eagle's medium (DMEM),containing 10% fetal calf serum, 4.5 g/L glucose, 100 U/mL penicillin,0.1 mg/mL streptomycin and 4 mM glutamine at 37° C. in a 5% CO₂humidified atmosphere. The cells were seeded on coverslip coated withpoly-ornithin and laminin at a density of 7.5×10⁴ cells per cm², 1 daybefore proceeding to the transfection. As shown in FIG. 13, only afaible, non-specific expression of some green fluorescence may bedetected in COS7—cells transfected with phuDCXpromoEGFP1 vector.

Transfection of the Cell Cultures with the phuDCXpromoEGFP1 Vector

Transfection was performed using 3 μg of phuDCXpromoEGFP1 vector in thepresence of 5 μl of the cationic lipid reagent Metafectene (BiontexLaboratories, Germany) in a final volume of 1 mL according to themanufacturers protocol. Two days after the end of the transfection, thecultures were examined for the expression of the EGFP reporter geneusing an Olympus IX70 inverted-fluorescent microscope. Nuclearcounterstaining was performed with 4′6′diamidino-2-phenylindoledihydrochloride hydrate at 0.25 μg/μL (DAPI, Sigma, Germany).

As documented in FIG. 13, the human DCX regulatory sequences areactivated to drive the expression of the EGFP reporter gene specificallyin neuronal restricted/determined cells.

EXAMPLE IV Analysis of transgenic mouse lines expressing the EGFPreporter gene under the control of the human DCX regulatory sequence

Generation of Transgenic Mouse Lines Expressing the EGFP Reporter GeneUnder the Control of the Human DCX Regulatory Sequence

For the generation of transgenic mouse lines, 100 micrograms ofphuDCXpromoEGFP1 vector was digested using the restriction enzymes AflIIand XhoI. A DNA fragment of approximately 4.5 kilobase pairs, SequenceID no:5, bearing the human DCX regulatory sequence, the EGFP gene and aSV40 poly adenylation signal, was obtained by agarose gelelectrophoresis followed by an extraction using the Geneclean kit (Bio101, Carlsbad, Calif., USA). A solution of. 10 mM Tris; 0.1 mM EDTAcontaining the purified the DNA fragment at 1 ng/microL was injectedinto the pronucleus of B6C3 μl mouse (Harland) embryos at the 1-cellstage. The surviving embryos were transferred the same day into theoviduct of 0.5 day post coitum pseudopregnant CD-1 recipient mice. Theprotocol for the generation of transgenic mouse lines is schematized inFIG. 14.

Mice bearing the huDCXpromoEGFP1 transgene were mated with C57BL/6Ncrlmice (Charles River Laboratories) to expand the colony.

Identification of the huDCXpromoEGFP1 Transgenic Mice

An approximately 5 mm tail piece was cut from the mice in order to gaingenomic DNA. The DNA was purified from the piece of tail using theDNeasy Tissue kit (Qiagen). PCR amplification was performed on mousegenomic DNA to detect the presence of the EGFP reporter gene. The PCRwas performed using the Amplitaq polymerase (Roche) and the followingoligonucleotide: oligo no.3: AAG TTC ATC TGC ACC ACC GGC (SEQ ID NO. 11)and oligo no.4: CTT TAC TTG TAC AGC TCG TCC (SEQ ID NO.: 12).

The following PCR protocol was used: 94° C. 2 minutes 1 cycle 94° C. 45seconds 30 cycles 59° C. 45 seconds 72° C. 2 minutes 72° C. 8 minutes 1cycle

The PCR amplification product was analysed by electrophoresis in agarosegel. Transgenic mice can be identified by the presence of anapproximately 600 base pairs PCR product.

Analysis of the huDCXpromoEGFP1 Transgene Expression

One month-old transgenic mouse from the line 299 and the line 303 weredeeply anesthetized and perfused transcardially with 4% paraformaldehydein 100 mM phosphate buffer, pH 7.4. The brains were dissected, immersedovernight in fixative, and transferred to 30% sucrose/100 mM phosphatebuffer, pH 7.4 for at least 48 hours. Brains were cut into 40 μnsagittal sections using a sliding microtome. Sections were stored at−20° C. in cryoprotectant solution until staining (25% v/v glycerol, 25%v/v ethylene glycol, and 0.05M phosphate buffer, pH 7.4).

For direct observation of the transgene expression pattern, sectionswere counterstained with 4′6′diamidino-2-phenylindole dihydrochloridehydrate at 0.25 μg/μL (DAPI, Sigma, Germany) in Tris-buffered saline(TBS: 0.1M Tris-HCl pH 7.4/0.9% NaCl) for 10 minutes. The DAPI staininglabels every nucleus of the section. The sections were washed twice for10 minutes in TBS and mounted on gelatin-coated glass slides. The slideswere coverslipped using Prolong (Molecular Probes, Eugene, U.S.A.).Documentation was performed using a Leica DMR microscope (LeicaMikroskopie und Systeme GmbH, Germany) equipped with a Spot digitalcamera (Diagnostic Instrument Inc. U.S.A.).

For immuno-histological analysis, sections were blocked in Tris-bufferedsaline (TBS: 0.1M Tris-HCl pH 7.4/0.9% NaCl) containing 3% donkey serumand 0.1% Triton-X 100 (TBS-DS-TX) for 30 min, followed incubation withprimary antibodies in TBS-DS-TX for 48 hours at 4° C. The followingprimary antibody dilutions were used: goat anti-DCX C-18 (1:500, SantaCruz Labs, Santa Cruz, USA) and rabbit anti-GFAP (1:1000, Dako,Danemark). The sections were then rinsed in TBS three times for 10minutes, and then incubated with secondary antibodies in TBS-DS-TX for 2hours. The following fluorochrome-conjugated secondary antibodies wereused: Alexa fluor 568 donkey anti-goat and Alexa fluor 568 goatanti-rabbit antibodies (4 micrograms/mL, Molecular Probes, Eugene,U.S.A.). Nuclear counterstain was performed with 0.5 microM TO-PRO-3(Molecular Probes, Eugene, U.S.A.) for 10 minutes. After several washesin TBS, sections were mounted on gelatin-coated glass slides andcoverslipped using Prolong (Molecular Probes, Eugene, U.S.A.).

Analysis was performed using a confocal microscope (TCS-NT, LeicaMicrosystems, Bensheim, Germany) equipped with a 40×PL APO oil objective(1.25 NA) and a pinhole setting that corresponded to a thickness of thefocal plane of less than 2 μm.

From the mouse embryos that were microinjected with the DNA fragmentencoding the EGFP reporter gene under the control of the human DCXregulatory sequence, 101 mice survived and developed into a maturemouse. PCR Screening of the genomic DNA isolated from the tail of themouse revealed that 8 animals were transgenic, 5 males and 3 females.The pattern of the EGFP reporter gene expression in the CNS was analyzedby observing brain sections from 1 month-old huDCXpromoEGFP1 transgenicmice from line 299 and from line 303 under a microscope forfluorescence. The high expression of the EGFP reporter was observed inthe dentate gyrus of the hippocampal formation, in the subventricularzone, in the rostral migratory stream and in the olfactory bulb (FIG.15). These regions are known to contain neuronal-restricted precursorcells and doublecortin expressing cells. Therefore, the expression ofthe EGFP reporter gene occurs in the relevant regions expected from theuse of the human DCX regulatory sequence.

Immunohistology was performed on brain sections from huDCXpromoEGFP1transgenic mice of the line 303 to define what type of cell expressedthe DCX reporter gene. FIG. 16 shows a co-localization of the endogenousdoublecortin expression in the olfactory bulb with the expression of theEGFP reporter gene. We also observed an absence of co-localizationwithin astrocytes, here detected with an ant-GFAP antibody, and theexpression of the EGFP reporter gene. Therefore the EGFP reporter geneis expressed in neuronal-restricted precursor cells expressing theendogenous doublecortin and not is not expressed in other cell types,like astrocytes.

EXAMPLE V Enrichment of Cells Expressing a Fluorescent Gene (EGFP orDsRed2) Derived from an Animal as Described in Example IV byFACS-Sorting

Brain tissue from transgenic mice of different developmental stages,comprising embryonic, postnatal and adult stages, are removed andtransferred into 4° C. DPBS (PAN, Germany) with or without 4.5 g/Lglucose (DPBS-glu solution). Overlying meninges and blood vessel areremoved as much as possible and the brain tissue is cut in small pieceswith a scalpel. Alternatively, specific brain regions, such ashippocampus, olfactory bulb, lateral ventricle wall, striatum,cerebellum or cortex are dissected. The tissue is washed in DPBS orDPBS-glu in order to rinse off the excess of blood and resuspended inPPD-solution composed of 0.01% Papain (Worthington Biochemicals,England), 0.1% dispase II (Roche, Germany), 0.01% DNase I (WorthingtonBiochemicals, England) 12.4 mM MgSO₄ in Hank's Balanced Salt Solution(HBSS, PAN, Germany) without Ca⁺⁺/Mg⁺⁺ and digested for 30 to 40 minutesat 37° C. with gentle trituration every 5 to 10 minutes. The cellsuspension is then centrifuged at 200×g for 5 minutes, wash two timeswith HBSS without Ca⁺⁺/Mg⁺⁺ and resuspended in 1 ml of HBSS withoutCa⁺⁺/Mg⁺⁺. The cell suspension is passed through a 30 μm cell-strainer(Becton-Dickinson, Germany).

The cell suspension is further sorted using a FACS Vantage flowcytometer equipped with a cell sorter (Becton-Dickinson, Germany) usingthe CELLQuest software. The cells population is analyzed using the lightforward and right-angle (side) scatter, the EGFP fluorescence through a530±15 nm bandpass filter and the DsRed fluorescence through a 575±13 nmbandpass filter, as they traverse the beam of an argon ion laser (488 nm100 mW). Additionally, dead cells could be excluded using substanceslabeling cells with damaged membranes, for example using a solution ofpropidium iodide at 10 μg/ml. Cells derived from a wild type mice can beuse to define the background fluorescence level. A sorting error levelunder 5%, resulting from false sorting or the presence ofdoublet/multiplex of cells composed of positive and negative cells, isexpected and acceptable. FIG. 28 illustrated sorted cell populationsobtained from dissociated brains of neonatal huDCXpromoDsRed2 transgenicmice. FACS-sorting of these dissociated brains for the presence ofDsRed2 fluorescence, resulted in a population of cells in which morethan 99% of the cells expressed the DsRed2 reporter gene as visualizeddirectly after sorting with an Olympus IX70 inverted-fluorescencemicroscope. Similarly, sorting for the absence of fluorescence in cellsof the dissociated brains resulted in a cell population in which lessthan 1% of the cells were expressing the DsRed2 reporter gene asvisualized directly after sorting with an Olympus IX70inverted-fluorescence microscope.

EXAMPLE VI Cells Expressing a Fluorescent Gene (EGFP or DsRed2) Derivedfrom an Animal as Described in Example IV Enriched by FACS YieldsDoublecortin-Positive Cells

Sorted viable cells from EXAMPLE V (above) are cultivated intoNeurobasal medium (Gibco BRL, Germany) supplemented with B27 (Gibco BRL,Germany), 5% fetal calf serum (PAN, Germany), 2 mM L-glutamine (PAN,Germany), 100 U/ml penicillin, 0.1 mg/ml streptomycin (PAN, Germany)into 12 wells/plate at different densities, e.g. 1×10⁵ cells per welland ml of medium, over glass coverslips pre-coated sequentially with asolution of 250 μg/ml poly-ornithin followed by a solution of 5 μg/mllaminin. Following different periods in culture, e.g. 1 day to 7 days,cells are fixed using a phosphate-buffered 4% paraformaldehyde solutionpH 7.4 (4% w/v paraformaldehyde, 100 mM NaH₂PO₄, 0.4 mM CaCl₂, 50 mMsucrose) for 30 minutes at room temperature. Sample are washed two timeswith DPBS (PAN, Germany) for 10 minutes at room temperature and blockedusing a fish-skin gelatin containing solution (0.1M Tris-HCl pH 7.5,0.15M NaCl, 1% bovine serum albumin, 0.2% Teleostean gelatin (Sigma,Germany), 0.1% Triton X-100 (Sigma, Germany)) for one hour at roomtemperature. The fish-skin gelatin containing solution is also used forthe antibody dilutions and the washing steps. The specimens areincubated overnight at 4° C. with the primary antibodies at thefollowing dilutions: goat anti-DCX C-18 1:100 to 1:1000 (Santa CruzLabs, USA); mouse anti-galactocerebrosides 1:500 (Chemicon, USA); rabbitant-GFAP 1:1000 (Dako, Danemark); rabbit anti-Ki67 1:500 (Novocastra,UK); rabbit anti-nestin 1:200 (Chemicon, USA); mouse anti-nestin 1:200(Pharmingen International, USA); mouse anti-βIII-tubulin 1:500 (clone5G8, Promega, USA); mouse anti-βIII-tubulin 1:500 (clone TUJ1, Babco,USA). The secondary fluochrome-conjugated antibodies are diluted 1:500(donkey anti-mouse or rabbit or goat, Dianova, Germany). Nuclearcounterstaining is performed with 4′,6′-diamidino-2-phenylindoledihydrochloride hydrate at 0.25 μg/μl (Dapi, Sigma, Germany). Followingthe last wash, the samples are briefly rinsed with PBS and mounted onslides using Prolong (Molecular Probes, The Netherlands). In cases whereantigens are sensitive to detergents, e.g. galactocerebrosides, TritonX-100 is omitted from the fish-skin gelatin containing solution.

Dissociated cells from neonatal transgenic mice expressing the EGFPreporter gene under the control of the neural nestin promoter(Kawaguchi, Mol Cell Neurosci 17 (2001), 259-273) can be used as aFACS-sorting control in a similar manner as described in the EXAMPLE V.Cells expressing nestin (in this transgenic model=expressing EGFP)constitute the population of neural stem cells. Upon differentiation,these cells have the potential to generate the three major types ofcells found in the central nervous system, namely neurons, astrocytesand oligodendrocytes. The cell population sorted for the presence of theEGFP reporter protein should have the potential in the experimentdescribed above to become neurons, astrocytes and oligodendrocytes. Thecell population sorted for the absence of EGFP reporter protein has alsothis potential, since it can be constitute from precursors cells forneurons and glia that are already more differentiated than the stemcells population and therefore do not express nestin (EGFP) anymore.Therefore, the two populations (positive and negative) of sorted cellsfor the expression of EGFP under the control of the neural nestinpromoter should generate mixed population of neurons and glia upondifferentiation.

FIG. 29 illustrates an immunostaining for doublecortin performed on cellcultures originating from dissociated brains of neonatalhuDCXpromoDsRed2 transgenic mice and from neonatal nestin-EGFPtransgenic mice that were FACS-sorted for the presence/absence offluorescent reporter protein. The cultures were fixed after one day ofculture in Neurobasal medium (Gibco BRL, Germany) supplemented with B27(Gibco BRL, Germany), 5% fetal calf serum (PAN, Germany), 2 mML-glutamine (PAN, Germany), 100 U/ml penicillin, 0.1 mg/ml streptomycin(PAN, Germany) into 12 wells/plate over glass coverslips pre-coatedsequentially with a solution of 250 μg/ml poly-ornithin followed by asolution of 5 μg/ml laminin, as described above. FIG. 29 documents thatFACS-sorting single cells suspension of dissociated brains from neonatalhuDCXpromoDsRed2 transgenic mice for cells expressing the reporter geneDsRed2 resulted in an enrichment of the population of cells expressingdoublecortin. FACS-sorting of the cells not-expressing the reporter geneDsRed2 resulted in a culture with very few doublecortin expressing cellsafter 1 day in culture. FIG. 29 also documents that FACS-sorting of asingle cells suspension of dissociated brains from neonatal nestin-EGFPtransgenic mice for cells expressing the reporter gene EGFP did notresult in an enrichment of the population of cells expressingdoublecortin as compared to the cells sorted for the absence of EGFPexpression.

EXAMPLE VII Cells Expressing a Fluorescent Reporter Gene (EGFP orDsRed2) Derived from an Animal as Defined in EXAMPLE IV enriched by FACSYields on Neuronal Precursors-Enriched Cultures

Sorted viable cells from EXAMPLE V (above) are cultivated intoNeurobasal medium (Gibco BRL, Germany) supplemented with B27 (Gibco BRL,Germany), 5% fetal calf serum (PAN, Germany), 2 mM L-glutamine (PAN,Germany), 100 U/ml penicillin, 0.1 mg/ml streptomycin (PAN, Germany)into 12 wells/plate at different densities, e.g. 1×10⁵ cells per welland ml of medium, over glass coverslips pre-coated sequentially with asolution of 250 μg/ml poly-ornithin followed by a solution of 5 μg/mllaminin. Following different periods in culture, e.g. 1 day to 7 days,cells are fixed using a phosphate-buffered 4% paraformaldehyde solutionpH 7.4 (4% w/v paraformaldehyde, 100 mM NaH₂PO₄, 0.4 mM CaCl₂, 50 mMsucrose) for 30 minutes at room temperature. Sample are washed two timeswith DPBS (PAN, Germany) for 10 minutes at room temperature and blockedusing a fish-skin gelatin containing solution (0.1M Tris-HCl pH 7.5,0.15M NaCl, 1% bovine serum albumin, 0.2% Teleostean gelatin (Sigma,Germany), 0.1% Triton X-100 (Sigma, Germany)) for one hour at roomtemperature. The same fish-skin gelatin containing solution is used forthe antibody dilutions and the washing steps. The specimens areincubated overnight at 4° C. with the primary antibodies at thefollowing dilutions: goat anti-DCX C-18 1:100 to 1:1000 (Santa CruzLabs, USA); mouse anti-galactocerebrosides 1:500 (Chemicon, USA); rabbitanti-GFAP 1:1000 (Dako, Danemark); rabbit anti-Ki67 1:500 (Novocastra,UK); rabbit anti-nestin 1:200 (Chemicon, USA); mouse anti-nestin 1:200(Pharmingen International, USA); mouse anti-βIII-tubulin 1:500 (clone5G8, Promega, USA); mouse anti-βIII-tubulin 1:500 (clone TUJ1, Babco,USA). The secondary fluochrome-conjugated antibodies are diluted 1:500(donkey anti-mouse or rabbit or goat, Dianova, Germany). Nuclearcounterstaining is performed with 4′,6′-diamidino-2-phenylindoledihydrochloride hydrate at 0.25 μg/μl (Dapi, Sigma, Germany). Followingthe last wash, the samples are briefly rinsed with PBS and mounted onslides using Prolong (Molecular Probes, The Netherlands). In cases whereantigens are sensitive to detergents, e.g. galactocerebrosides, TritonX-100 is omitted from the fish-skin gelatin containing solution.

Dissociated cells from neonatal transgenic mice expressing the EGFPreporter gene under the control of the neural nestin promoter(Kawaguchi, Mol Cell Neurosci 17 (2001), 259-273) can be used as aFACS-sorting control in a similar manner as described in the EXAMPLE V.Cells expressing nestin (in this transgenic model=expressing EGFP)constitute the population of neural stem cells. Upon differentiation,these cells have the potential to generate the three major types ofcells found in the central nervous system, namely neurons, astrocytesand oligodendrocytes. The cell population sorted for the presence of theEGFP reporter protein should have the potential in the experimentdescribed above to become neurons, astrocytes and oligodendrocytes. Thecell population sorted for the absence of EGFP reporter protein has alsothis potential, since it can be constitute from precursors cells forneurons and glia that are already more differentiated than the stemcells population and therefore do not express nestin (EGFP) anymore.Therefore, the two populations (positive and negative) of sorted cellsfor the expression of EGFP under the control of the neural nestinpromoter should generate mixed population of neurons and glia upondifferentiation.

Immunodetection of βIII-tubulin performed on cell cultures originatingfrom dissociated brains of neonatal huDCXpromoDsRed2 transgenic mice andfrom neonatal nestin-EGFP transgenic mice that are FACS-sorted for thepresence/absence of fluorescent reporter protein. The cultures are fixedafter one day of culture in Neurobasal medium (Gibco BRL, Germany)supplemented with B27 (Gibco BRL, Germany), 5% fetal calf serum (PAN,Germany), 2 mM L-glutamine (PAN, Germany), 100 U/ml penicillin, 0.1mg/ml streptomycin (PAN, Germany) into 12 wells/plate over glasscoverslips pre-coated sequentially with a solution of 250 μg/mlpoly-ornithin followed by a solution of 5 μg/ml laminin, as describedabove. FACS-sorting of single cells suspension of dissociated brainsfrom neonatal huDCXpromoDsRed2 transgenic mice for cells expressing thereporter gene DsRed2 results in an enrichment of the population of cellsexpressing βIII-tubulin. Since βIII-tubulin is expressed in neuronalprecursors, FACS-sorting of cells expressing the huDCXpromoDsRed2 vectoris an efficient mean generate from a mixed cell population a cellculture enriched in neuronal precursors. FACS-sorting of the cellsnot-expressing the reporter gene DsRed2 results in a culture containingvery few of βIII-tubulin expressing cells after 1 day in culture.FACS-sorting of a single cells suspension of dissociated brains fromneonatal nestin-EGFP transgenic mice for cells expressing the reportergene EGFP does not result in an enrichment of the population of cellsexpressing βIII-tubulin as compared to the culture of cells FACS-sortedfor the absence of EGFP reporter gene expression.

EXAMPLE VIII Enrichment of EGFP-Positive Cells by Transfection andFACS-Sorting

Cell Culture Preparation

Embryonic rat, mouse and chicken forebrain cultures:

Embryonic mouse, rat or chicken forebrain cultures are prepared asfollows: pregnant rats such Sprague-Dawley, Whistar, Fisher 344, orpregnant mice, such as C57/bl6 are sacrificed at gestation day 10.5 bypentobarbital overdose, their fetuses removed, decapitated, and theirbrains dissected, Special care is taken not to contaminate withmeningeal tissue and skull osteoid. For chicken brain cultures, theforebrain is dissected from freshly decapitated embryos after 6, 8, 10or 12 days of gestation. In each case, the tissue is collected in 4° C.DPBS (PAN, Germany) with 4.5 g/L glucose (Merck, Germany) (DPBS/glu).The tissue is enzymatically treated in PPD-solution containing 0.01%Papain (Worthington Biochemicals, England), 0.1% dispase II (Boehringer,Germany), 0.01% DNase I (Worthington Biochemicals, England) and 12.4 mMMgSO₄ in HBSS (PAN, Germany) without Mg⁺⁺/Ca⁺⁺ (PAA, Germany) anddigested for 20 min at 37° C. with gentle trituration every 5 min. Thecell suspension is then centrifuged at 200 g for 5 Min, wash 2× withHBSS —Ca/—Mg and resuspended in 2 ml HBSS —Ca/—Mg. Cells are dissociatedby triturating sequentially 20/10/5, through serially-narrowed glasspasteur pipette, and resuspended in NB medium (Gibco BRL, Germany)supplemented with B27 (Gibco BRL, Germany) (NB/B27), 5% FCS (PAN,Germany), 2 mM L-glutamine (PAN, Germany), 100 μg/ml penicillin/0.1mg/mL streptomycin (PAN, Germany). Cells are plated on poly-ornithine(250 μg/ml) and laminin (5 μg/ml) coated 12 well plates at differentcell densities (10E5 to 10E7 cells/well and ml) and grown for two toseven days.

Adult Cultures

Brains from mice, such as C57/bl6 or rats, such as Sprague-Dawley,Whistar, Fisher 344, elder than 1 month are removed after scarifying,Overlying meninges and blood vessels are removed and the brain tissue iscut in small pieces with a scalpel. Alternatively, specific brainregions, such as hippocampus, olfactory bulb, lateral ventricle wall,striatum, cerebellum or cortex are dissected. The tissue is washed inDPBS/glu in order to rinse off excess blood and resuspended inPPD-solution containing 0.01% Papain (Worthington Biochemicals,England), 0.1% dispase II (Boehringer, Germany), 0.01% DNase I(Worthington Biochemicals, England) and 12.4 mM MgSO₄ in HBSS (PAN,Germany) without Mg⁺⁺/Ca⁺⁺ (PAA, Germany) and digested for 30 to 40 minat 37° C. with gentle trituration every 10 min. The cell suspension isthen centrifuged at 200 g for 5 Min, wash 2× with HBSS —Ca/—Mg andresuspended in 2 ml HBSS —Ca/—Mg. Cells are dissociated by trituratingsequentially 20/10/5, through serially-narrowed glass pasteur pipetteand passed through a 30 μm cell strainer (Becton-Dickinson). andresuspended in NB medium (Gibco BRL, Germany) supplemented with B27(Gibco BRL, Germany) (NB/B27), 5% FCS (PAN, Germany), 2 mM L-glutamine(PAN, Germany), 100 μg/ml penicillin/0.1 mg/mL streptomycin (PAN,Germany). Cells are plated on poly-ornithine (250 μg/ml) and laminin (5μg/ml) coated 12 well plates at different cell densities (10E5 to 10E7cells/well and ml) and grown for two to seven days.

Explant Cultures:

Alternatively to dissociated monolayer cultures, tissue explants is alsoused for transfection experiments. Here, adult mouse, rat or human braintissue, such as hippocampus, ventricle wall or olfactory bulb areisolated and dissected as described aboe and cut in small pieces with ascalpel. Additional dissection in 200 μm pieces is performedautomatically using a McIllwain tissue chopper. Explants are than platedonto poly-ornithine (250 μg/ml) and laminin (5 μg/ml) coated 35 mmFalcon dishes in NB medium (Gibco BRL, Germany) supplemented with B27(Gibco BRL, Germany) (NB/B27), 5% FCS (PAN, Germany), 2 mM L-glutamine(PAN, Germany), 100 μg/ml penicillin/0.1 mg/mL streptomycin (PAN,Germany).

Transfection Methods:

Liposomal transfection: Cells derived from different sources, such asembryonic or adult, such as chicken, mouse rat and human are plated asdescribed above. Two to seven days after plating, medium is exchanged totransfection medium comprising of NB medium (Gibco BRL, Germany)supplemented with B27 (Gibco BRL, Germany) (NB/B27), 2 mM L-glutamine(PAN, Germany) to remove serum and Pen/Strep for 1 h. 3 μg DNA (SEQ IDNO. 7; EGFP-vector under the control of DCX regulatory sequence) and 5μl of kationic lipid reagent (Metafectene) are each diluted in 50 μltransfection medium. Dilutions of DNA and Metafectene are gently mixed,mixture is incubated at room temperature for 20 min before pipetting onthe cells in the 12-well-plate. Transfection lasts 8 to 10 hours, thenthe supernatent is exchanged by fresh, antibiotic and serum containingmedium. Imaging for EGFP is performed 12-38 hrs after transfection,using an OlympusIX70 microskope with epifluorescence optics.Particel-mediated gene transfer: Alternatively to liposomal-basedtransfection, genes can be introduced into different kind of cells byparticle-mediated delivery, using a Biolistics particle delivery system(Bio-Rad PDS 1000). Explant cultures are prepared as described above.After an overnight incubation, the brain explants are well adherent onthe dish. Medium is removed during, and readded after gene delivery. Forgene delivery, gold particles (0.6 or 1 μm, 50 ml of 60 mg/ml, Bio-Rad)are coated with 5 μg of plasmid DNA, such as (SEQ ID NO. 7; EGFP-vectorunder the control of DCX regulatory sequence) for 1 h, after which theparticles are collected by centrifugation, washed and resuspended in 50μl 100% ethanol. A total of 8 μl of gold particle/DNA suspension isadded on a sterile macrocarrier disc (Bio-Rad) and the ethanol isevaporated. The macrocarrier is then mounted in a Biolistic partibledelivery system and the samples are placed in 5 to 10 cm distance fromthe stopping screen. Helium with pressure of 1500 psi, and a rupturepressure of 1000-1200 psi is provided by using a 1100 psi rupture disk.Bombardment is performed at chamber vacuum of 20-25 Hg.

Alternatively to liposomal-based or particle-mediated gene deliverymethods, viral mediated gene delivery using vectors such as adenoviral,adeno-associated viral, lenti viral, or retroviral vectors can beconceived to deliver a marker-gene under the control of the DCXregulatory sequence. Different cell types including embryonic or adult,mouse, rat, chicken or human may be used for gene delivery of the EGFPreporter gene under the control of the human DCX promoter using thedifferent viral vectors. Using an adenoviral vector at a concentrationof 10 pfu/cell, roughly 20% of cultures forebrain cells express EGFP 3days after infection. At 1000 pfu/cells, over 50% of cells express EGFP.

FACS-Sorting: Cells are dissociated by using Accutase (PAA) and loadedwith 10 μg/ml propidium iodide (PI) and passed through a 30 μm cellstrainer (Becton-Dickinson). Cells are analyzed and sorted using a FACSVantage flow cytometer/cell sorter (Becton-Dickinson) equipped withCELLQuest software). Cells (2×10E6 cells/ml) are analyzed by lightforward and right-angle (side) scatter, PI fluorescence, and EGFPfluorescence through a 510±20 nm bandpass filter, as they traverse thebeam of an with an argon ion laser (488 nm 100 mW). Dead cells areexcluded by gating on forward and side scatter, and by eliminatingpropidium iodide-positive events. Cells that were not transfected areused to set the background fluorescence. A false positive rate of0.02±0.05% is accepted so as to ensure an adequate yield.

EXAMPLE IX Cells expressing a fluorescent gene (EGFP) derived from antransfection experiment enriched by FACS yields Doublecortin-positivecells

Viable cells form EXAMPLE VIII (above) are sorted into NB medium (GibcoBRL, Germany) supplemented with B27 (Gibco BRL, Germany) (NB/B27), 5%FCS (PAN, Germany), 2 mM L-glutamine (PAN, Germany), 100 μg/mlpenicillin/0.1 mg/mL streptomycin (PAN, Germany) at a speed of 3000events/s. Sorted cells are plated on poly-ornithine (250 μg/ml) andlaminin (5 μg/ml) coated glass coverslips in 12 well plates at differentce'l densities (10E5 to 10E7 cells/well and ml and grown for two toseven days. Cells are fixed with phosphate-buffered 4% prewarmed 37° C.paraformaldehyde pH 7.4 (4% w/v paraformaldehyde, 100 mM NaH₂PO₄, 0.4 mMCaCl₂, 50 mM sucrose) for 30 min and processed for immunohistochemistry.Following 30 min of fixation at room temperature with phosphate-buffered4% paraformaldehyde, samples are blocked for a minimum of 1 hour in fishskin gelatin buffer (0.1M Tris-HCl pH 7.5, 0.15M NaCl, 1% bovine serumalbumin, 0.2% Teleostean gelatin (Sigma, Germany), 0.1% Triton X-100) atroom temperature. The specimens are incubated overnight at 4° C. withthe primary antibodies at the following dilutions: goat anti-DCX C-18(1:500, Santa Cruz Labs, Santa Cruz, USA); mouse anti-galactocerebroside1:500 (Chemicon, USA); rabbit anti-GFAP 1:1000 (Dako, Danemark); rabbitanti-Ki67 1:500 (Novocastra, UK); rabbit anti-nestin 1:200 (Chemicon,USA); mouse anti-nestin 1:200 (Pharmingen International, USA); mouseanti-βIII-tubulin 1:500 (clone 5G8, Promega, USA); mouseanti-βIII-tubulin 1:500 (clone TUJ1, Babco, USA). The secondaryfluorochrome-conjugated antibodies are diluted 1:500 (donkey anti-mouseor rabbit, Dianova, Germany). All antibody dilutions and washes areperformed with the fish skin gelatin buffer. Nuclear counterstaining isperformed with 4′,6′-diamidino-2-phenylindole dihydrochloride hydrate at0.25 μg/μl (DAPI, Sigma, Germany). Following the last wash, the samplesare briefly rinsed with PBS and mounted on slides using Prolong(Molecular Probes, The Netherlands). In cases where antigens aresensitive to detergents (GaIC), Triton X-100 is omitted from the fishskin gelatin buffer.

Dissociated cells are sorted into one of two fractions, a EGFP negativeand a EGFP positive one. Sorted cells are cultured and analyzed byimmunohistochemistry as described above. 2 days after sorting, mostcells of the EGFP positive fraction continued to express EGFP andimmunostaining for doublecortin revealed that more than 90% of cellsexpress doublecortin. In contrast, very few of the cells from the EGFPnegative fraction expresses doublecortin.

Two days after cell sorting, less than 5% of the cells derived from theEGFP positive population expresses the neuroectodermal progenitor markerNestin, non of the cells express the glial marker GFAP or theoligodendroglial marker GaIC, however, more than 90% of the cellsexpressed βIII tubulin, a neuronal marker that is turned on early duringneuronal differentiation. Doublecortin expressing cells co-express 13111tubulin. Very few of the cells derived from the EGFP negative fraction,doublecortin is expressed. However, we find that less than 1% of thecells express Nestin, more than 30% of the cells express GFAP, no GaICstaining can be detected, but 10% of the cells express βIII tubulin.These are likely to be mature neurons, since some cells express also theneuronal marker MAP2.

Seven days after sorting and plating, the EGFP signal in cells derivedfrom the EGFP positive fraction decreases and is only hardly detectable.At the same time, doublecortin expression decreases in the culture asdetermined by immunstaining. None of the cells in these cultures inpositive for Nestin, but most cells do now express βIII tubulin. Many ofthese cells (more than 20%) express MAP2 and GAP-43, marker for maturingneurons. In cultures derived from the EGFP negative fraction, the numberof GFAP positive glial cells increases probably due to glial growth,whereas the number of neurons does not change.

EXAMPLE X Transplantation Experiments Using Cells Enriched in EGFP-DCXExpressing Cells

Cells can be derived either from an transgenic animal as described inExample IV or from transfected cells as described in Example III.

Transplantations are conducted according to protocols previouslyestablished. Single cell suspensions are prepared for transplantation byFACS sorting as described above and sorted into HBSS. Cells areresuspended in HBSS at a concentration of 100,000 cells/μl. The specificinjection parameters need to be determined depending on the animalmodel, but initially cells are injected at specific sites with eachinfusion site receiving 0.5 μl (50,000 cells). A single volumetricpressure injection of the cell suspension into the brain is performedusing a digitally controlled microinjector via a pulled-glassmicropipette with tip diameter of approximately 100 μm.

Grafting of human or mouse cells into rodents requires immunosuppressionin order to prevent rejection of grafts by the host. This is achieved bycontinuous treatment with cyclosporine or FK506.

Animal Models

Alzheimer's Disease (AD). The cholinergic neurons of the cholinergicforebrain system are significantly affected in AD and their progressivecell death leads to cognitive impairment and dementia. Animals withdiscrete lesions of the forebrain cholinergic system exhibit similardeficits in learning and memory tasks. Lesion are performed oncholinergic cells in the medial septum, diagonal band and nucleusbasalis by intraventricular injection of a specific neurotoxin(192-IgG-Saporin). This neurotoxin is selectively taken up bycholinergic neurons and leads to cell death within 7 days afterapplication. When the lesion is complete the levels of cholinergicactivity in the cortex and hippocampus are reduced to less than 20% ofpreoperative values. In brief, intraventricular infusions of theimmunotoxin is performed by injecting 3.5 μg 192IgG-saporin in a finalvolume of 5 μl saline over a period of 5 min into the right lateralventricle. Transplantations are performed about 2 weeks after lesion inorder to test the ability of cells to replace the lost cholinergicneurons and to improve function in learning tasks.

Parkinson's Disease. A mouse model that is widely used as an animalmodel of PD is based on the systemic application of the compound1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for review see,(Kaakkola and Teravainen (1990); Langston (1985). Major steps in theexpression of neurotoxicity involve the conversion of MPTP to the toxicagent 1-methyl-4-phenylpyridinium ion (MPP+) by type B monoamine oxidase(MAO-B) in glial cells, the specific uptake of MPP+ into nigro-striataldopaminergic neurons and the intraneuronal accumulation of MPP+. Itinduces selectively cell death of the dopaminergic cells in thesubstantia nigra and produces severe motor deficits. Briefly, C57BL/6mice (8 weeks-old) receive a single s.c. injection of 40 mg/kg MPTP.This regimen has been shown typically to induce a reproducible 80%striatal dopamine depletion in C57BL/6 mice (Chan (1997)) within 7 daysafter injection. One week after inducing Parkinson-like symptoms in micecells are grafted into the striatum to improve dopamine release in thisstructure, and the substantia nigra for reconstruction of thenigro-striatal projection.

Stroke: the medial cerebral artery occlusion (MCAO) model. Briefly, maleWistar rats weighing 250-270 g are anesthetized with 4% isoflurane in amixture of 70% N₂O and 30% O₂ and intubated orotracheally for mechanicalventilation. A thermostatically regulated heating lamp and pad are usedto maintain body temperature at 37° C. Local cortical blood flow in eachhemisphere is monitored in the cerebral artery by laser-dopplerflowmetry. After exposure of the right carotid bifurcation and ligationof the branches of the internal carotid artery (ICA) under the operatingmicroscope, the external carotid artery is ligated and cut distal to thesuperior thyreoid artery. Then, a silicone-coated 4-0 nylon monofilamentis introduced into the ECA and gently advanced through the ICA until itstip occludes the origin of the MCA. By this, local cortical blood flowin the MCA territory drops to about 20% of baseline. The endovascularsuture remains in place until reperfusion is allowed by withdrawal ofthe filament. Transplantation sites for cells include the basal gangliaand subcortical white matter (ischemic core) and the overlying cortex(ischemic penumbra) of the ischemic hemisphere.

Morphology

Transplantation of cells into animal models of neurological diseasesprovide information about the integration and survival of grafted cells,their differentiation potential under pathological conditions and theircapability to functionally replace dying neurons. For morphologicalevaluation the brains are removed at predetermined time points aftergrafting. Specific antibody immunohistochemical staining will determinethe number and distribution of grafted cells. In combination with cellmarker for neurons, astrocytes and oligodendrocytes, the differentiationfate for cells is determined via multiple-immunofluorescence andconfocal laser microscopy. When necessary, characterization ofindividual neuronal phenotypes will be conducted using transmitterspecific antibodies.

Also envisaged are grafting experiments from other species, e.g., mousecells may be grafted into, e.g., rat (brains) and migration behaviour,survival and/or differentiation of said grafted cells may be tested.

Tissue preparation. Animals are killed at different time points aftergrafting with an overdose of anesthetics and perfused transcardiallywith 4% paraformaldehyde in phosphate buffer. The brains are removed,stored in the fixative overnight, and then transferred to 30% sucrose.For free-floating immunohistochemistry, 40-μm sections are cut on asliding microtome and stored at −20° C. in cryoprotectant solution.

Quantitative stereology. Sections through the grafted regions arestained immunohistochemically for the protein that is expressed underthe control of the DCX regulatory sequence.

EXAMPLE XI Deletion- and Transfection Studies Generation of DeletionMutants from the Human DCX Regulatory Sequence in Order to Define theHuman DCX Minimal Promoter and its Regulatory Elements

Generation of Deletions Mutants:

Using the subcloned human DCX regulatory sequence, for example thephuDCXpromoEGFP1 vector, it is possible to design deletion protocolsusing restriction enzymes. By digesting the vector with appropriaterestriction enzymes, it is possible to cut apart a segment of the humanDCX regulatory sequence. The remaining piece of the vector can possesscompatible ends for recircularization using T4 DNA ligase or may need tobe first blunt-ended. In order to generate a fragment of DNA withcompatible blunt ends, one may make use of the DNA polymerase I Klenowfragment in the presence of an excess of free dNTPs.

Alternatively, segments of the human DCX regulatory sequence can beamplified by PCR and subcloned in the promoterless EGFP-N1 vector, asdescribed in the Example II. This strategy allows for a more preciseselection of the regions to be analyzed, without the requirement ofsites for restriction enzymes. Several of these amplified fragments canbe easily subcloned in tandem in front of a reporter gene, such as theEGFP reporter gene.

Analysis of the Expression Promoting Activity of the Human DCXRegulatory Sequence and its Sub-Regions:

The expression promoting activity of the human DCX regulatory sequence,as well as from the various deletion mutants, can be assess bytransfecting neuronal-restricted progenitor cells. These cells can begained from the dissociation of forebrains from embryonic day 10.5 to12.5 mouse embryo. The cells suspensions obtained from these embryonicforebrains contain several cell-types, including neuronal-restrictedprecursor cells. Parallel transfections of these dissociated forebraincultures can be performed using the EGFP reporter gene under the controlof an ubiquitous promoter, such as CMV, under the control of the humanDCX regulatory sequence or under the control of any fragments of thehuman DCX regulatory sequence. Comparison of the levels of EGFP reportergene expression, as visualized under a microscope for fluorescence,allows for the analysis of the expression promoting activity of thesequence controlling the expression of the EGFP reporter gene.Alternatively, the transfected cells can be analyzed by FACS, allowingfor a rapid quantification of the percentage of cells expressing theEGFP reporter gene as well as the relative intensity of expression.

The neuronal-restricted precursor cells can also be gained by the use ofhuDCXpromoEGFP1 transgenic mice. Dissociation of regions of the CNSbearing neurogenesis will provide a cell suspension containing severalcell types, including neuronal-restricted precursor cells. This cellsuspension can be divided into EGFP reporter expressing cells and EGFPreporter non-expressing cells fractions by FACS-sorting using thefluorescent signal of the EGFP reporter protein as the sorting criteria.The EGFP positive cells would correspond to neuronal-restrictedprecursor cells, and the EGFP negative cells to all the other celltypes. These two populations can then be transfected, for example, withthe DsRed2 fluorescent reporter gene under the control of the human DCXregulatory sequence and under the control of fragments of the human DCXregulatory sequence.

The cell-specificity of regulatory sequences is often the result ofexpression inducing elements, that induce the expression of the gene ina specific desired cell type, and expression inhibitory elements, thatblocks the expression of the gene in cell types other then the desiredcell type. The use of the two sorted cells fractions permits for theidentification of inducing and inhibitory elements. Analysis of theexpression of the DsRed2 reporter gene in the transfected EGFP positivecells, allows to determined if the regulatory sequence is able to inducethe expression of the DsRed2 reporter gene in cells which areneuronal-restricted, revealing the presence of sufficientexpression-inducing elements. Analysis of the expression of the DsRed2reporter gene in the transfected EGFP negative cells allows to determineif some expression inhibitory elements have been deleted. In this case,DsRed2 expression would be observed in EGFP negative cells.

The use of established cell lines, such as COS7 cells, can also be usedto assess the deletion of expression inhibitory elements in the humanDCX regulatory sequence, resulting in the expression of the reportergene in cells other than neuronal-restricted precursor cells.

EXAMPLE XII Putative Transcription Factor Binding Sites in the DCXRegulatory Sequences

For sequence analysis the following programs were applied: fasta20u66,version 2.0u66 available at Biology Workbench of the San DiegoSupercomputer Center (http://workbench.sdsc.edu) for alignment;MatInspector, version 6.2.1, available at Genomatix Software GmbH(Munich, Germany, www.genomatix.de) for analysis of transcription factorbinding sites (Quandt (1995) Nucleic Acids Research 23, 4878-4884).

The analysis of the SEQ ID NO:1 revealed putative binding sites for Bm-2(position 1985-2001; 2774-2790), NeuroD1 (position 1821-1833), E2F-1(positions 1831-1845; 2675-2689; 2761-2775), E2F-2 (positions2309-2323), Smad3 (position 1397-1405), Smad4 (position 1397-1405) (FIG.17). The analysis of the SEQ ID NO: 2 revealed a similar pattern likebinding sites for Bm-2 (positions 153-166; 2136-2152), Bm-3 (position926-942), NeuroD1 (positions 178-190; 1154-1166; 2468-2480), E2F-1(positions 196-210; 561-575; 1136-1150; 1616-1630; 1800-1814), E2F-2(position 2578-2592) and Fast1 (positions 574-588; 646-660) (FIG. 17).

EXAMPLE XIII The DCX Regulatory Sequence is Active Specific in NeuronalDetermined Cells In Vitro

Cells and Cell Culture

If not otherwise cited, solutions for cell culture were obtained fromPAN Biotech GmbH (Aidenbach, Germany), plastic culture vessels from TPP(Trasadingen, Switzerland). HEK293 cells (Clontech, Heidelberg, Germany)and COS7 cells (ATCC U.S.A., # CRL-1651) were maintained in Dulbecco'smodified Eagles's medium adjusted to contain 10% fetal calf serum, 100U/ml penicillin, 0.1 mg/ml streptomycin, 4 mM L-glutamine, 1.5 g/lsodium bicarbonate and 4.5 g/l glucose at 37° C. in a 5% CO₂.containinghumidified atmosphere. CTX TNA2 (rat astrocyte line, ECACC #98102213)were maintained in DMEM adjusted to contain 10% fetal calf serum, 100U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 1.5 g/lsodium bicarbonate and 1 mM sodium pyruvate at 37° C. in a 5% CO₂containing humidified atmosphere. N20.1, a mouse oligodendrocyte line,was kindly provided by Anthony T. Campagnoni, University of California,Los Angeles, USA. For proliferation, N20.1 cells were maintained inHAM's-F12/DMEM 1:1 supplemented with 2.4 g/l glucose, 0.18 g/lL-Glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 22.5 mg/lGentamicin at 33° C. in a 5% CO₂ containing humidified atmosphere. Inorder to differentiate this line into a oligodendroglial phenotype,cells were maintained in the same medium, but at 39° C. for 7 days.D283Med (ATCC U.S.A. #HTB-185) and Neuro2A (ATCC U.S.A. #CCL-131) weremaintained in Minimal essential medium (Eagle) with Earle's BSS adjustedto contain 10% fetal calf serum, 100 U/ml penicillin, 0.1 mg/mlstreptomycin, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 1 mM sodiumpyruvate and 0.1 mM non-essential amino acids at 37° C. in a 5%CO₂.containing humidified atmosphere. Cells were seeded on plasticculture plates at a density of 2.5×10⁴ cells per cm² one day beforeproceeding to the transfection. Before seeding D283Med cells, culturedishes were coated with poly-ornithin (250 μg/ml) and laminin (15μg/ml). 1 day after transfection, cells were reseeded on glasscoverslips coated with poly-ornithin (25.0 μg/ml) and laminin (15 μg/ml)at a density of 7.5×10⁴ cells per cm². Mouse embryonic forebrain (MEF)cultures were prepared as follows: pregnant C57BI/6Ncrl mouse females(Charles River Laboratories, Sulzfeld, Germany) were sacrificed bycervical dislocation. The uteri were removed an immersed in ice-coldDulbecco's phosphate buffered saline solution (DBPS). Embryonic day 10.5to 12.5 embryos were released from the uteri, the forebrains weredissected and separated from surrounding tissues. The dissectedforebrains were washed once in ice-cold DPBS, transferred to a petridish and dissociated mechanically with a scalpel blade. The resultingpreparation was washed in DPBS. Following 5 min centrifugation at 120×g,the pellet was resuspended in PPD-solution containing 0.01% Papain(Worthington Biochemicals, England), 0.1% dispase II (Roche, Germany),0.01% DNase I (Worthington Biochemicals, England) and 12.4 mM MgSO₄ inHBSS (PAN, Germany) without Mg⁺⁺/Ca⁺⁺ (PAA, Germany) and incubated for30 to 40 min at 37° C. The cell solution was triturated every 10 minthrough a pipette. Dissociated cells were collected by 5 mincentrifugation at 120×g. The pellet was resuspended in serum-freeneurobasal medium (Gibco BRL, Germany) and washed three times. Finally,the cell preparation was resuspended in Neurobasal medium supplementedwith B27 (Gibco BRL, Germany), 2 mM L-glutamine, 100 U/ml penicillin,0.1 mg/ml streptomycin, 5% fetal calf serum, seeded on coverslips coatedwith poly-ornithin (250 μg/ml) and laminin (15 μg/ml) at a density of2.5×10⁵ cells per cm² and maintained for four days at 37° C. in 5%CO₂.containing humidified atmosphere before transfection.

Transfections:

Transfections of cell lines were performed in 10 Cm² culture dishes(6-well-plates) using 3 μg of plasmid (phuDCXpromoEGFP1 orphuDCXpromoDsRed2) per dish in the presence of 5 μl Metafectene (BiontexLaboratories, Munich, Germany) in a final volume of 2 ml according tothe manufacturer's protocol. Medium was exchanged after 12 hours. Oneday after the end of transfection, cells were trypsinized and reseededon Poly-Ornithin/Laminin coated glass coverslips for another 24 h beforeimmunohistological analysis. MEF cells were transfected directly on thecoated glass coverslips they had been seeded on to form a differentiatedculture. Transfections of MEF cells were performed using 1.2 μg ofplasmid per 4 cm² culture surface in the presence of 3 μl Metafectene ina final volume of 1 ml according to the manufacturer's protocol. Mediumwas exchanged after 12 hours, cells were incubated for another 4 daysunder cell culture conditions before immunohistological analysis. Afterthe end of transfection, all cultures were examined for the expressionof the EGFP or DsRed2 reporter gene using an Olympus IX70 invertedfluorescent microscope. To prepare cell extracts of transfected celllines or transfected MEF primary cultures, cells were seeded andtransfected on 10-cm² dishes and harvested directly from the culturevessel with a cell scraper.

Immunostainings

Following 30 min of fixation at room temperature with phosphate-buffered4% paraformaldehyde, samples were blocked for a minimum of 1 hour infish skin gelatin buffer (0.1M Tris-HCl pH 7.5, 0.15M NaCl, 1% bovineserum albumin, 0.2% Teleostean gelatin (Sigma-Aldrich, Taufkirchen,Germany), 0.1% Triton X-100) at room temperature. The specimens wereincubated overnight at 4° C. with the primary antibodies at thefollowing dilutions: goat anti-doublecortin C18 1:1000 (Santa CruzBiotechnology, Santa Cruz, Calif., USA); rabbit anti-galactocerebroside1:250 (AB142, Chemicon, Temecula, Calif., USA); rabbit anti-GFAP 1:1000(Dako, Glostrup, Denmark); rabbit anti-Ki67 1:500 (Novocastra, Newcastleupon Tyne, UK); mouse anti-nestin 1:500 (BD Biosciences Pharmingen, SanDiego, Calif., USA); mouse anti-βIII-tubulin 1:500 (clone 5G8, Promega,Madison, Wis., USA); rabbit anti-neurofilament 200 (Chemicon, Temecula,Calif., USA); mouse anti-map-2a/b (Sigma-Aldrich, Taufkirchen, Germany).The secondary fluorochrome-conjugated antibodies were diluted 1:500(Rhodamine Red™-X-conjugated-, Cy™5-conjugated-,Fluorescein(FITC)-conjugated donkey anti-mouse, rabbit and goat anti IgGfrom dianova GmbH, Hamburg, Germany). All antibody dilutions and washeswere performed with the fish skin gelatin buffer. Nuclearcounterstaining was performed with 4′,6′-diamidino-2-phenylindoledihydrochloride hydrate at 0.25 μg/μl (DAPI, Sigma-Aldrich, Taufkirchen,Germany) or TO-PRO-3 (Molecular Probes, Eugene, Oreg., USA). Followingthe last wash, the samples were briefly rinsed with PBS and mounted onslides using Fluoromount (Southern Biotechnology Associates, Birmingham,Ala., USA). In cases where antigens were sensitive to detergents, TritonX-100 was omitted from the fish skin gelatin buffer.

Western Blot Analysis

To prepare protein extracts of transfected cell lines or transfected MEFprimary cultures, cells were seeded and transfected on O-cm² dishes.Cells were harvested from the culture vessel in phosphate bufferedsaline using a cell scraper and collected by 5 min centrifugation at200×g. Proteins were extracted by homogenizing the cell pellet inSUB-buffer (0.5% sodium-dodecyl-sulfate, 8M urea, 2% β-mercaptoethanol),passing the suspension at least 5 times through a 20 gauge needle fittedto a 2 ml-plastic-syringe and separating the lysate from debris bycentrifugation at 10.000×g for 10 min. Concentration of total protein inthe supernatant was assayed using Bradford's method. 5 μg of totalprotein per lane was run on a 12.5% SDS-PAGE gel at 2 mA/cm and blottedat 2.5 mA/cm² for 0.7 h on a nitrocellulose membrane (0.45 μm pore size,Protran BA 85; Schleicher+Schuell, Dassel, Germany). Blocking of themembrane, incubation with antibodies and washing steps were performed infish-skin-gelatine western buffer (20 mM Tris-HCl pH 7.3, 0.9% NaCl, 1%fish skin gelatine, 0.1% Tween-20). To detect doublecortin, EGFP oractin immunologically, the membrane was blocked for 2 hours at roomtemperature, probed with anti-DCX (goat IgG, 1:1000 dilution, SantaCruz, Santa Cruz, Calif., USA), anti-EGFP (goat IgG, 1:1000 dilution,Rockland, Gilbertsville, Pa., USA) or anti-actin (rabbit IgG, 1:5000dilution, Sigma, Taufkirchen, Germany) antibody overnight at 4° C. Blotswere rinsed and incubated with a horseradish peroxidase-conjugateddonkey anti goat IgG (1:50.000 dilution, Sigma, Taufkirchen, Germany) oranti rabbit IgG secondary antibody (1:50.000 dilution, Dianova, Hamburg,Germany) for 2 hours at room temperature and rinsed before performingECL detection of protein-antibody conjugates (ECL-Plus Western BlottingDetection System, Amersham-Pharmacia, Freiburg, Germany) according tothe manufacturer's protocol.

Quantitative Analysis

Immunostainings of cultured cells were examined using confocal lasermicroscopy. In order to determine the ratio of cells expressing acertain marker or a reporter protein, five randomly picked visual fieldswere examined under a magnification of 200×. First, the total cellnumber in the field was determined according to a nuclear counterstain,then the number of cells positive for the marker or the transgene wasdetermined. In order to detect colabeling of endogenous markers orcolabelling of marker and reporter, cells expressing marker or reporterwere picked randomly and examined for co-expression with the othermarker. For this purpose, a magnification of 400× was used, a totalnumber of 40 cells positive for the reporter or the marker was examinedin each paradigm.

Specific activity of the human DCX regulatory sequence in cells ofneuronal lineage. To test the activity of the SEQ ID NO.:1 as promoter,the expression vector phuDCXpromoEGFP1 (SEQ ID NO: 7) (FIG. 8A) wastransiently transfected into different cell types. HEK293 and COS7 cellswere used as examples for non-neural cells, glial lineage restrictedcells were represented by the astrocytic cell line CTX TNA2 and by theoligodendroglial line N20.1, mixed neural population by the mouseneuroblastoma cell line Neuro-2a and by human medulloblastoma D283 Medcells, and for a most physiological relevant cell type, primary mouseembryonic forebrain cells of embryonic day 10.5 to 12.5 (MEF) were used.Expression was analyzed by Western-blot analysis and qualitative andquantitative fluorescence microscopy. Expression of EGFP driven by SEQID NO:1 was not or barely detected in the non-neural cells HEK293 andCOS7 and not in the CTX TNA2 and the N20.1, weakly detectable in theneural cell lines D283 Med and Neuro-2a, and strongly detectable in MEFcells (FIG. 18). The few non-neuronal cells expressing. EGFP under thehuman DCX regulatory sequence expressed EGFP only at low levels,compared to expression driven by the CMV enhancer in these cell lines orcompared to expression driven by the human DCX regulatory sequence inthe neuronal Neuro-2a and MEF cells (FIG. 18). The activity of the DCXpromoter fragment correlated well with expression of endogenous DCX, asdemonstrated by Western-blot analysis. Control transfections withpEGFP-N1 to drive EGFP expression via the ubiquitous CMV promoterdemonstrated that all cell types were transfectable and expressed EGFP(FIG. 18). These transfected cells expressed EGFP at high levels (FIG.18). However, transfection rates varied between the different celltypes, so that HEK293, COS7 and Neuro-2a cells showed transfectionefficiencies from 6% to 14%, whereas CTX TNA2, D283 and primary MEF hadtransfection efficiencies below 3% (FIG. 19). Calculating the relativepromoter activity coefficient by (% cells expressing phuDCXpromoEGFP1divided by % cells expressing pEGFP-N1)×100 indicates that in up to30.6% of transfected MEF cells, the human DCX regulatory sequence isactive (FIG. 19). Lower specific activity was found in Neuro-2a and D283cells (approx. 7 to 12%), and no activity was found in the non-neuronalcell lines HEK293, CTX TNA2 and N20.1. COS7 cells activated the humanDCX regulatory sequence rarely and weakly. As compared to the strongexpression observed in neuronal cells, this level is considerednon-significant. In summary, these experiments suggest that the humanDCX regulatory sequence is predominantly active in proliferative cellsof neuronal lineage.

The human DCX regulatory sequence is predominantly active in youngneuronal determined cells.

The next sets of experiments were targeted towards the question,whether, in transient transfection experiments, the SEQ ID NO: 1 has asimilar activity compared to the endogenous DCX promoter. For that, wemade use of the MEF cells. This culture system is comprised of differentcell types of the developing nervous system.

The immunocytochemical analysis revealed a low percentage of nestinpositive neural stem cells (1.17%) and KI67 positive proliferating cells(2.24%) (FIG. 20). The vast majority of cells expressed markers foryoung neurons, such as DCX (16.43%), Map2ab (16.08%) and βtubIII(24.31%), few cells expressed the mature neuronal marker NF200 (2.34%)(FIG. 20). GFAP was expressed in 12.87% of the cells, and theoligodendrocytic marker GaIC was detected in only 0.32% of the cells.DCX colocalized with cells expressing Map2ab (92.5%), βtubIII (72.5%)and NF200 (5%), not with the neural stem cell marker nestin and not withcells expressing the mature neuronal marker NF200, the astrocytic markerGFAP or the oligodendrocytic marker GaIC (FIG. 21).

In cells expressing EGFP after transient transfection with SEQ ID NO.: 7a similar pattern was observed (FIG. 22, FIG. 23). None of the EGFPpositive cells were immunoreactive for nestin nor for the glial markerGaIC, a minor percentage of EGFP expressing cells stained for the glialmarker GFAP (2.5%), 25% of EGFP expressing cells were positive for K167,67.5% for Map2ab, 25% for tubIII, 22.5% for NF200. Most importantly, thevast majority of EGFP expressing cells were immunoreactive for DCX(82.5%) (FIG. 23), indicating that the human DCX regulatory sequenceactivity correlates with the endogenous DCX gene expression. In additionto the marker expression that suggests a young neuronal phenotype forthe EGFP expressing cells, the cells display a morphology characteristicfor young neuronal or neuronal precursor cells, indicated by a smallcell soma and a bipolar to oligopolar neurite pattern (FIG. 22).

Deletions in the DCX promoter define regions required for expression andfor specificity.

The sequence analysis of the human DCX regulatory sequence definedseveral possible important regions (FIG. 10). To analyze the relevanceof some of these regions, several deletion mutations were constructedand tested for their activity in MEF cells (FIG. 24). Accordingly,Region 2 comprises a critical part of the regulatory sequence (FIG. 25).

EXAMPLE XIV DCX Expression as a Marker for NeuronalTrans-differentiation

Retinal pigment epithelium (RPE) cells of higher mammals are fullydifferentiated epithelial cells that do not express neuronal markers invivo (Zhao, Int. Rev. Cytol. 171 (1997), 225-266). Substantial evidence,however, exists for de- or trans-differentiation of RPE cells. In vivo,experimentally induced retinal detachment causes focal proliferation ofRPE cells at the site of detachment in a number of mammalian species(Anderson, Invest. Ophthalmol. Vis. Sci. 21 (1981), 10-16). In thisparadigm, RPE cells round up, loose their apical processes and divide toform a three to four cells thick layer. In vitro, de- andtrans-differentiation of mammalian RPE cells is well documented. Primarycultures of RPE cells loose differentiation markers such as pigmentationand the differentiation markers RPE10 and RPE65 (Neill. Invest.Ophthalmol. Vis. Sci. 34 (1993), 453462; Hamel, J. Neurosci. Res. 34(1993), 414-425). Neuronal trans-differentiation was found in human RPEcultures by expression of the early neuronal marker beta III-tubulin andin a minor population of cultured human RPE cells that express theneuron-specific enolase and the neuronal marker neurofilament 200(Vinores, Exp. Eye Res. 60 (1995), 385400). In addition, the mRNA forthe neuronal specific microtubule-associated protein MAP 1B is inducedin human cultured RPE cells (Esser, Invest. Ophthalmol. Vis. Sci. 38(1997), 2852-2856). This example demonstrates DCX expression intrans-differentiating RPE cells.

Adult Long Evans rats were briefly sedated and sacrificed bydecapitation. Eyes were removed and stored in ice cold DPBS (PAN,Germany). Eyes were dissected by first opening the eye shortly behindthe border of the sclera and the rest of the eye. For preparation ofciliary body derived stem cells (CB), the frontal part of the eye wasdissected clean of the surrounding tissues, including the vitreus, RPEcells, iris and neural retina and pieces of the ciliary body weredigested in PPD solution (0.01% Papain (Worthington Biochemicals, USA),0.1% Dispase II (Boehringer Mannheim, Germany), 0.01% Dnase I(Worthington Biochemicals, USA) and 149 mg MgSo₄*7H₂O in Hank's BalancedSalt Solution w/o Ca²⁺/Mg²⁺ (PAN, Germany) for 100 ml). For preparationof RPE cells, the neural retina was taken out of the eye cup and then 2%Dispase II solution was applied to the cup. The cups were collected in96 well plates and incubated at 37° C. for 30 min. After that the RPEcells were gently washed from the underlying choroid tissue andharvested in ice-cold PBS. After a 15 min incubation step in PPDsolution, the cells were processed equally to the CB derived cells.Typically, the tissue of 10 eyes was digested and the PPD solutioncontaining the cells were triturated every 10 min. Dissociated cellswere collected by centrifugation (188 rcf) and resuspended in 5 ml coldDMEM/F12. Cells were washed three times with thorough trituration.Finally, cells were plated at 1×10⁵ cells/2 ml in Neurobasal (NB) mediumsupplemented with B27, 0.1 g/L Penicilline/Streptavidine, 2 mML-Glutamine, 20 ng/ml EGF, 20 ng/ml FGF-2 and 2 μg/ml Heparin plus 1%fetal calf serum (FCS, PAA, Germany). Cells were seeded in 6 well plates(each well at 35 mm) and cultures were maintained at 37° C. in anincubator with 95% air, 5% CO₂. Single cells began to form sphereswithin 2 to 3 days in culture and continued to grow in mass and numberover the next weeks. Half of the medium was changed every 4 days. Forpassaging of cells, the culture medium containing the floating sphereswas collected in a 15 ml centrifuge tube and centrifuged at 188 rcf for5 min. The pellet was resuspended in 200 μl of Accutase™ and triturated5 times. Additionally the cell suspension was placed at 37° C. for 10min. After dissociation, the cells were again triturated, centrifuged at188 rcf and resuspended in NB/B27 medium. To visualize proliferation inimmunohistochemical studies, the thymidin-analogue Bromodeoxyuridin(BrdU) was used. Cells after passage #3 were supplemented with 10 μMBrdU in NB/B27 for 24 hrs prior to seeding on coated glass cover slips.During the time of differentiation (7 days), medium was changed every 3days, but no new BrdU was added.

Dissociated cells from the adult ciliary margin (CB) or retinal pigmentepithelium (RPE) were placed in culture and analyzed for theirproliferative potential. When first plated after dissociation, thecultures were composed of single, mostly pigmented cells thatoccasionally appeared in small clusters, probably due to a not completedissociation during the preparative process. After an initial growthphase of approximately 34 days, the cells started to form aggregates, socalled spheres. The very same process has been detected foradult-derived stem cells from other neurogenic regions of the centralnervous system, e.g. the hippocampus (HC) or the subventricular zone(SVZ). The spheres grew in size over the next 4 weeks. Spheres grew bycell proliferation as indicated by BrdU incorporation and a quantitativeanalysis of the cell number showed that the cultures expanded over time.In addition, a clonal analysis revealed that both CB and RPE derivedcells are able to clonally expand under limited dilution conditions andtherefore indicate a similarity to known stem cell cultures from e.g.the HC or the SVZ. In cultures derived from the CB, the cell numberinitially increased by three-fold during the first two weeks. Then, cellproliferation ceased and the number of cells reached a plateau,suggesting that the generated cells did survive in culture. The increasein cell number was slightly delayed in RPE derived cells, but basicallyshowed the same kinetics as the CB cells and eventually also reached aplateau with a cell count that could not be increased by longercultivation. Both CB and RPE derived cells could be kept in culture forup to 12 weeks (or 6 passages) before a change in the morphology of thecells indicated an arrest of growth and proliferation. Dissociated andpassaged cells readily reformed spheres with similar growth kineticscompared to primary spheres. Cells were tested for expression of theneural stem cells markers Nestin and Musashi, the proneural gene Pax6,the neuronal precursor marker DCX and early neuronal marker βIIITubulin. CB and RPE derived cells were grown under proliferationconditions (NB/B27 medium with EGF/FGF/Heparin and 1% FCS). RT-PCRanalysis was performed and showed that these cells express the neuronalprecursor marker DCX (FIG. 26), indicating that indeed, adult and fullydifferentiated CB and RPE cells can transdifferentiate into a neuronalcell. Approximately 8% of cells were found to be immunoreactive for DCXby immunostaining (FIG. 27). In addition, βIII Tubulin was expressed inup to 15% of cells (FIG. 27). Suprisingly, βIII Tubulin was expressed intwo types of cells: one with flat epithelial appearance, and one with aneuronal morphology including bi- or pluripolar structure, elongatingprocesses. Doublelabeling for DCX illustrates that DCX identifies theβIII Tubulin subpopulation with neuronal morphology, indicating that DCXis indeed a marker for neuronal restriction and neuronaldetermination/differentiation in these cells and suggesting that DCX isa reliable indicator for neuronal differentiation and neuronaltrans-differentiation.

EXAMPLE XV Enrichment of Neuronal Trans-Differentiating Cells Expressinga Fluorescent Gene (EGFP) Under the Regulatory Sequence of the Human DCXGene as Described Herein by FACS-Sorting

RPE cells are prepared from eyes of huDCXpromoEGFP1 transgenic mice ofembryonic, postnatal and adult stages, and cultured as described inExample XIV. Cells are dissociated by using Accutase (PAA) and loadedwith 10 μ/ml propidium iodide (PI) and passed through a 30 micron cellstrainer (Becton-Dickinson). Cells are analyzed and sorted using a FACSVantage flow cytometer/cell sorter (Becton-Dickinson) equipped withCELLQuest software. Cells (2×10E6 cells/ml) are analyzed by lightforward and right-angle (side) scatter, PI fluorescence, and EGFPfluorescence through a 510±20 nm bandpass filter, as they traverse thebeam of an with an argon ion laser (488 nm 100 mW). Dead cells areexcluded by gating on forward and side scatter, and by eliminatingpropidium iodide-positive events. Cells from wild type animals are usedto set the background fluorescence. A false positive rate of 0.02±0.05%is accepted so as to ensure an adequate yield.

Viable cells are sorted into NB medium (Gibco BRL, Germany) supplementedwith B27 (Gibco BRL, Germany) (NB/B27), 5% FCS (PAN, Germany), 2 mML-glutamine (PAN, Germany), 100 U/ml penicillin/0,1 mg/ml streptomycin(PAN, Germany) at a speed of 3000 events/s. Sorted cells are plated onpoly-ornithine (250 μg/ml) and laminin (5 μg/ml) coated glass coverslipsin 12 well plates at different cell densities (10E5 to 10E7 cells/welland ml and grown for two to seven days. Cells are fixed withphosphate-buffered 4% prewarmed 37° C. paraformaldehyde pH 7.4 (4% w/vparaformaldehyde, 100 mM NaH₂PO₄, 0.4 mM CaCl₂, 50 mM sucrose) for 30min and processed for immunohistochemistry. Following 30 min of fixationat room temperature with phosphate-buffered 4% paraformaldehyde, samplesare blocked for a minimum of 1 hour in fish skin gelatin buffer (0.1MTris-HCl pH 7.5, 0.15M NaCl, 1% bovine serum albumin, 0.2% Teleosteangelatin (Sigma, Germany), 0.1% Triton X-100) at room temperature. Thespecimens are incubated overnight at 4° C. with the primary antibodiesat the following dilutions: goat anti-DCX C-18 (1:500, Santa Cruz Labs,Santa Cruz, USA); mouse anti-galactocerebroside 1:500 (Chemicon, USA);rabbit anti-GFAP 1:1000 (Dako, Danemark); rabbit anti-Ki67 1:500(Novocastra, UK); rabbit anti-nestin 1:200 (Chemicon, USA); mouseanti-nestin 1:200 (Pharmingen International, USA); mouseanti-βIII-tubulin 1:500 (clone 5G8, Promega, USA); mouseanti-pill-tubulin 1:500 (clone TUJ1, Babcd, USA). The secondaryfluorochrome-conjugated antibodies are diluted 1:500 (donkey anti-mouseor rabbit, Dianova, Germany). All antibody dilutions and washes areperformed with the fish skin gelatin buffer. Nuclear counterstaining isperformed with 4′,6′-diamidino-2-phenylindole dihydrochloride hydrate at0.25 μg/μl (DAPI, Sigma, Germany). Following the last wash, the samplesare briefly rinsed with PBS and mounted on slides using Prolong(Molecular Probes, The Netherlands). In cases where antigens aresensitive to detergents (GaIC), Triton X-100 is omitted from the fishskin gelatin buffer.

Dissociated cells are sorted into one of two fractions, a EGFP negativeand a EGFP positive one. Sorted cells are cultured and analyzed byimmunohistochemistry as described above. Two days after sorting, mostcells of the EGFP positive fraction continued to express EGFP andimmunostaining for DCX revealed that more than 90% of cells express DCX.In contrast, none of the cells from the EGFP negative fraction expressesDCX.

EXAMPLE XVI In Vivo Imaging of Adult Neurogenesis Using a TransgenicMouse Comprising SEQ ID NO: 26 (Human DCX RegulatorySequence—Luciferase)

In vivo imaging of physiological events by EGFP and/or luciferasereporter genes is becoming more and more interesting, since it allowsthe analysis of physiological and/or pathophysiological dynamic changesin a longitudinal and real time manner (Gambhir, Neoplasia 2 (1-2)(2000), 118-38; Wu, Mol. Ther. 4(4) (2001), 297-306). In this example,changes in the level of adult neurogenesis are detected in vivo byluminometric measurements.

Transgenic mice are generated as in the Examples above, but instead ofexpressing EGFP or DSRed2, the luciferase gene is placed under controlof the human DCX regulatory sequence as defined herein (see SEQ ID NO:26 and SEQ ID NO: 27). Coelenterazine (Hayward, Calif., USA), asubstrate for renilla luciferase, and D-luciferin firefly potassium salt(Xenogen, Calif., USA), the substrate for firefly luciferase, are used.The in vivo Imaging System (IVIS, Xenogen) consists of a cooled CCDcamera mounted on a light-tight specimen chamber (dark box), a cameracontroller, a camera cooling system, and a Windows computer system. Thetransgenic mouse is placed in the specimen chamber mounted with the CCDcamera cooled to −120° C., with a field of view set at 25 cm above thesample shelf. The photon emission is measured. The grey scalephotographic images and bioluminescent color images are superimposedusing the LIVINGIMAGE V. 2.11 software overlay (Xenogen) and IGOR imageanalysis software (V. 4.02 A, Wave Metrics, Lage Oswego, Oreg.). Aregion of interest (e.g. the head) is manually selected.

Mice are anesthetized by i.p injection of a ketamine/xylazine (4:1)solution. Mice are injected by tail-vein injection with 0.7 mg/kg bodyweight of coelenterazine or 150 mg/kg body weight D-luciferin andscanned with fifteen 1-min scans using the cooled CCD camera.

Bioluminescence is specifically detected in the two neurogenic regions,dentate gyrus of the hippocampus and the subventricularzone—rostramigratory stream—olfactory bulb axis. No other brain regionis bioluminescent, and so is the rest of the body, when scans from otherbody regions are performed. In the next set of experiments, neurogenesisin the adult brain is up-regulated by the experimental conditions suchas physical activity, administration of anti-depressants and ofexperimentally-induced seizures. Here the changes in bioluminescencecorrelate with the described changes in neurogenesis. There is a cleardecline in the level of bioluminescence in the dentate gyrus of thehippocampus with animals of increasing age. The decrease inbioluminescence can also be observed within one and the same animal in alongitudinal study. Similarly, the up-regulation of neurogenesis in thehippocampus after seizure can easily be detected by the elevated levelof bioluminescence.

EXAMPLE XVII Use of huDCXpromoEGFP1 Transgenic Mice to Study Variationin Neurogenesis Levels

The level of neurogenesis in the dentate gyrus has been reported todecrease during aging (Kuhn, J. Neurosci. 6 (1996), 2027-2033). UsinghuDCXpromoEGFP1 transgenic mice from EXample IV, the quantification ofEGFP fluorescence intensity in the neurogenic areas can serve as areporter of the level of neurogenesis in these regions.

For the study of the age-related decrease in neurogenesis occurring inthe dentate gyrus, mice of the huDCXpromoEGFP1 line 303 of 1, 2 and 12months of age were deeply anesthetized and perfused with 4%paraformaldehyde in 100 mM phosphate buffer pH 7.4. The brains weredissected, immersed overnight in fixative and transferred to 30%sucrose/100 mM phosphate buffer pH 7.4 for 24 hours. Brains were cutinto 40 μm sagittal sections using a sliding microtome. Sections werestored at −20° C. in cryoprotectant solution until use (25% v/vglycerol, 25% v/v ethylene glycol, 0.05M phosphate buffer pH 7.4).

For comparison of the fluorescence intensity between the transgenic miceof different ages, sections were put on microscope slides (SuperFrostPlus, Menzel-Gläser, Germany) and mounted using Fluoromount-G (SouthernBiotechnology Associates, Inc., USA). The fluorescence signal wasrecorded using a Leica microscope (Leica Mikroskopie und Systeme GmbH,Wetzlar, Germany) equipped with a Spot™ digital camera and software(Diagnostic Instrument, Inc., Sterling Heights, USA). The fluorescenceintensity was quantified by measuring the mean gray value of the pixelswithin the granular cells area of the dentate gyrus using the ImageJsoftware (Image J 1.29, National Institutes of Health, USA). The levelof fluorescence observed in the cortex of the 1 month-old animal wasused as the background fluorescence level and was subtracted from meangray values calculated for dentate gyri. FIG. 30 (Panels A to C)documents the levels of fluorescence detected in the dentate gyrus ofthe young versus the old huDCXpromoEGFP1 line 303 transgenic mice. Thepicture acquisitions were performed using the same illumination,exposure time and image post-processing for all transgenic mice,allowing therefore for a direct comparison. FIG. 30 documents asignificant decrease in the level of EGFP fluorescence detected in thedentate gyrus of the aged transgenic mice as compare to the levelsobserved in the young transgenic mice, hence confirming that the use ofhuDCXpromoEGFP1 transgenic mice provides an efficient mean to monitorthe variation in the levels of neurogenesis between individuals. FIG.30F presents the result of the quantification performed using ImageJ onthe dentate gyrus of four mice, i.e. the three presented in FIGS. 30A-Cand a fourth animal of 2 months of age. For every animal, 4 sectionscontaining a segment of dentate gyrus were quantified in order togenerate an average and a standard deviation. Quantificationdemonstrates that the reduction of neurogenesis observed during aging isaccompanied by a reduction of the EGFP fluorescent signal as a functionof aging, i.e. 1 month>2 months>12 months. Also, comparison of the twomice of 2 months of age demonstrate that the variation inter-individualin minimal.

Epileptic seizures have been reported to provoke an increase ofneurogenesis level in the dentate gyrus (Parent, J. Neurosci. 17 (1997),3727-3738). Using huDCXpromoEGFP1 transgenic mice, comparison of EGFPfluorescence intensities in the dentate gyrus of control animals andanimals in which epileptic seizures were experimentally induced allowsfor the measurement of variation of neurogenesis.

To this end, huDCXpromoEGFP1 transgenic mice of 3 months of age received1 mg of scopolamine per kilogram of body weight i.p. (Sigma, St. Louis,Mo., USA). Twenty minutes later, these animals were injected with 340 mgof pilocarpine per kilogram of body weight i.p. (Sigma, St. Louis, Mo.,USA). Seizure activity was monitored behaviorally, and after 120minutes, the seizures were terminated using 10 mg of diazepam perkilogram of body weight i.p. (Sigma, St. Louis, Mo., USA). Only micethat displayed continuous, convulsive seizure activity after pilocarpinetreatment were used in this experiment. Control mice received salineinjections in replacement to scopolamine and pilocarpine, butnevertheless received diazepam injections. All animals received food andwater ad libitum. The animals were processed for histological analysis 7days after the seizure induction. Animals were deeply anesthetized andperfused with 4% paraformaldehyde in 100 mM phosphate buffer pH. 7.4.The brains were dissected, immersed overnight in fixative andtransferred to 30% sucrose/100 mM phosphate buffer pH 7.4 for 24 hours.Brains were cut into 40 μm sagittal sections using a sliding microtome.Sections were stored at −20° C. in cryoprotectant solution until use(25% v/v glycerol, 25% v/v ethylene glycol, 0.05M phosphate buffer pH7.4).

For comparison of the fluorescence intensity between the transgenicmice, sections from animals of the same age were put on microscopeslides (SuperFrost Plus, Menzel-Gläser, Germany) and mounted usingFluoromount-G (Southern Biotechnology Associates Inc., USA). Thefluorescence signal was recorded using a Leica microscope (LeicaMikroskopie und Systeme GmbH, Wetzlar, Germany) equipped with a Spot™digital camera and software (Diagnostic Instrument Inc, SterlingHeights, USA). The fluorescence intensity was quantified by measuringthe mean gray value of the pixels within the granular cells area of thedentate gyrus using the ImageJ software (Image J. 1.29, NationalInstitutes of Health, USA). The level of fluorescence observed in thecortex of the control animal was used as the background fluorescencelevel and was subtracted from mean gray values calculated for dentategyri. FIGS. 30D-E documents an increase in the level of EGFPfluorescence detected in the dentate gyrus of the transgenic mice inwhich epileptic seizures have been experimentally-induced as compare tothe level observed in the control transgenic mice, confirming that theuse of huDCXpromoEGFP1 transgenic mice provides an efficient mean tomonitored the variation in the levels of neurogenesis betweenindividuals. FIG. 30F presents the result of the quantificationperformed using ImageJ on the dentate gyrus of the control mouse and theone in which seizures was experimentally induced. For every animal, 4sections containing a segment of dentate gyrus were quantified in orderto generate an average and a standard deviation. Quantification (FIG.30F) demonstrates that the increase in neurogenesis occurring in theanimal in which seizures have been experimentally induced is accompaniedby an increase of the EGFP fluorescent signal. Therefore, quantificationof the EGFP fluorescence signal in the huDCXpromoEGFP1 transgenic micemay easily be employed to quantify modulation in neurogenesis.

1-56. (canceled)
 57. A DNA segment comprising a regulatory sequenceisolated free of the complete DCX gene protein coding region, whereinthe regulatory sequence comprises a regulatory sequence selected fromthe group consisting of: (a) regulatory sequences comprising thenucleotide sequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, asshown in SEQ ID NO: 3 or as shown in SEQ ID NO: 4; (b) regulatorysequences comprising the nucleotide sequence contained in the insertionof clone DSM 15111 and obtainable by amplification using twooligonucleotides having the sequences indicated under SEQ ID NO: 9 andSEQ ID NO: 10; (c) regulatory sequences comprising at least onenucleotide sequence of SEQ ID NO: 1 from position 1166 to 1746, fromposition 1166 to 2049, from position 1785 to 1843 or from position 1953to 2775; (d) regulatory sequences comprising at least one nucleotidesequence of SEQ ID NO: 2 from position 529 to 1079, from position 529 to1390, from position 1118 to 1175 or from position 1291 to 2137; (e)regulatory sequences comprising at least a functional part of a sequenceof (a) to (d) and causing specific expression in neuronal determinedcells; (f) regulatory sequences comprising a nucleotide sequence whichis at least 75% identical to a sequence as defined in (a) to (d) orwhich comprises a nucleotide sequence which is at least 78% identical tothe nucleotide sequence as shown in SEQ ID NO: 1 from position 1166 to1746 or from position 1166 to 2049 or to the nucleotide sequence shownin SEQ ID NO: 2 from position 529 to 1079 or from position 529 to 1390,which comprises a nucleotide sequence which is at least 82% identical tothe nucleotide sequence as shown in SEQ ID NO: 1 from position 1785 to1843 or to the nucleotide sequence as shown in SEQ ID NO: 2 fromposition 1118 to 1175 or which comprises a nucleotide sequence which isat least 75% identical to the nucleotide sequence as shown in SEQ ID NO:1 from position 1953 to 2775 or to the nucleotide sequence as shown inSEQ ID NO: 2 from position 1291 to 2137; and (g) regulating sequencescomprising a nucleotide sequence which hybridizes with a complementarystrand of the regulatory sequence as defined in (a) to (f) for theearly, transient expression of a heterologous nucleotide sequence inproliferative neuronal determined cells.
 58. The DNA segment of claim57, wherein said DNA segment further comprises a heterologous nucleotidesequence to be expressed.
 59. The DNA segment of claim 57, wherein saidregulatory sequence is of human, mouse or rat origin.
 60. The DNAsegment of claim 58, wherein said heterologous nucleotide sequence to beexpressed is a gene selected from the group consisting of a marker gene,a receptor gene, an anti-apoptotic gene, a determination/differentiationgene, a gene capable of inducing and/or directing neuronal migration orguidance, a gene encoding a tag, a gene encoding for a trophic factor, agene encoding a surface protein, a gene encoding for a transcriptionfactor or a gene encoding an enzyme, or wherein said nucleotide sequenceto be expressed is an antisense sequence or encodes for a ribozyme or aninhibiting RNA molecule.
 61. The DNA segment of claim 60, wherein saidmarker or receptor gene is selected from the group consisting of GFP,EGFP, RFP, CFP, BFP, YFP, DsRed, f3-galactosidase, luciferase orchloramphenicol acetyltransferase.
 62. The DNA segment of claim 60,wherein said gene encoding a tag is selected from the group consistingof a His-Tag, glutathione, a Strep-tag, a Flag-tag, CBP, TAG-100, E2-tagand Z-tag.
 63. The DNA segment of claim 60, wherein said gene encoding asurface protein is CD24.
 64. The DNA segment of claim 60, wherein saidgene encoding for a trophic factor is selected from the group consistingof NGF, BDNF, PDGF, NT-3, NT-4, NT-5, VEGF, PEDF, EGF, FGF, IGF,cardiotrophin, erythropoietin, leptin, LIF, and TGF.
 65. The DNA segmentof claim 60, wherein said anti-apoptotic gene is selected from the groupconsisting of bcl-2, Brn-3a, PTEN and (an) anti-caspase(s).
 66. The DNAsegment of claim 60, wherein said determination/differentiation gene isthe dopaminergic determination factor Nurr1.
 67. The DNA segment ofclaim 60, wherein said gene capable of inducing and/or directingneuronal migration or guidance is selected from the group consisting ofnetrin, neuropilin, CXCR4, SDF-1, DCC, slit, robo, a semaphorin, aplexin family member, and an ephrin family member.
 68. The DNA segmentof claim 60, wherein said gene encoding a transcription factor isselected from the group consisting of NeuroD, BMP4, Nurr1 or ShcC. 69.The DNA segment of claim 60, wherein said gene encoding an enzyme isCRE.
 70. The DNA segment of claim 60, wherein said heterologousnucleotide sequence to be expressed is an antisense sequence, or encodesfor a ribozyme or an inhibiting RNA-molecule.
 71. The DNA segment ofclaim 70, wherein said inhibiting RNA is selected from the groupconsisting of RNAi, siRNA, shRNA and stRNA.
 72. The DNA segment of claim58, wherein said DNA segment is further defined as a recombinant vectorcomprising said a regulatory sequence and heterologous nucleotidesequences to be expressed positioned under the control of saidregulatory sequence.
 73. The DNA segment of claim 72, wherein saidvector is further defined as a virus.
 74. The DNA segment of claim 72,wherein said vector is further defined as vector is suitable for genetherapy or vaccination with a nucleic acid molecule.
 75. The DNA segmentof claim 57, wherein the regulatory sequence comprises the nucleotidesequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown inSEQ ID NO: 3 or as shown in SEQ ID NO:
 4. 76. The DNA segment of claim57, wherein the regulatory sequence comprises the nucleotide sequencecontained in the insertion of clone DSM 15111 and obtainable byamplification using two oligonucleotides having the sequences indicatedunder SEQ ID NO: 9 and SEQ ID NO:
 10. 77. The DNA segment of claim 57,wherein the regulatory sequence comprises at least one nucleotidesequence of SEQ ID NO: 1 from position 1166 to 1746, from position 1166to 2049, from position 1785 to 1843 or from position 1953 to
 2775. 78.The DNA segment of claim 57, wherein the regulatory sequence comprisesat least one nucleotide sequence of SEQ ID NO: 2 from position 529 to1079, from position 529 to 1390, from position 1118 to 1175 or fromposition 1291 to
 2137. 79. The DNA segment of claim 57, wherein theregulatory sequence comprises a nucleotide sequence which is at least75% identical to the regulatory sequences set forth in SEQ ID NOs 1through 4, or which comprises a nucleotide sequence which is at least78% identical to the nucleotide sequence as shown in SEQ ID NO: 1 fromposition 1166 to 1746 or from position 1166 to 2049 or to the nucleotidesequence shown in SEQ ID NO: 2 from position 529 to 1079 or fromposition 529 to 1390, which comprises a nucleotide sequence which is atleast 82% identical to the nucleotide sequence as shown in SEQ ID NO: 1from position 1785 to 1843 or to the nucleotide sequence as shown in SEQID NO: 2 from position 1118 to 1175 or which comprises a nucleotidesequence which is at least 75% identical to the nucleotide sequence asshown in SEQ ID NO: 1 from position 1953 to 2775 or to the nucleotidesequence as shown in SEQ ID NO: 2 from position 1291 to
 2137. 80. TheDNA segment of claim 57, wherein the regulatory sequence comprises anucleotide sequence which hybridizes with a complementary strand of theregulatory sequence as defined in claim 78 under stringent conditionsand which provided for the early, transient expression of a heterologousnucleotide sequence in proliferative neuronal determined cells.
 81. Ahost cell comprising a DNA segment in accordance with claim
 58. 82. Thehost cell of claim 81, wherein said DNA segment is positioned such thatthe heterologous nucleotide sequence is expressible by said cell. 83.The host cell of claim 81, whereis said cell is further defined asneuronal cell, an ES-cell, a germ cell, a cultured cell or a primarycell.
 84. The host cell of claim 80, further defined as a neuronaldetermined cell.
 85. A method of expressing a heterologous nucleotidesequence in a host cell comprising obtaining a host cell in accordancewith claim 82 and culturing said host cell in a manner effective toachieve the expression of said heterologous nucleotide sequence.
 86. Themethod of claim 85, wherein the host cell is a neuronal determined celland the method is further defined as a method for the early expressionof the heterologous nucleotide sequence in the neuronal determined cell.87. The method of claim 85, wherein said host cell comprises a neuronalcell, an ES-cell, a germ cell, a cultured cell or a primary cell. 88.The method of claim 85, wherein the host cell is further defined as aneuronal determined cell.
 89. A method of expressing a nucleotidesequence to be expressed comprising positioning such a nucleotidesequence under the control of a regulatory sequence as defined by claim57 and expressing such nucleotide sequence in a host cell.
 90. A methodof preparing a genetically modified host cell comprising obtaining aregulatory sequence in accordance with claim 57 and introducing saidsequence into a selected host cell to provide a genetically modifiedhost cell.
 91. A method for the generation of a non-human transgenicanimal comprising the step of introducing a regulatory sequence asdefined in claim 1 into an ES-cell or a germ cell.
 92. A non-humantransgenic animal which comprises host cells as defined by claim
 81. 93.The non-human transgenic animal of claim 92, wherein said transgenicanimal is a mouse or a rat.
 94. The non-human transgenic animal of claim92, which comprises a nucleotide sequence under the control of saidregulatory sequence which encodes for GFP, EGFP, RFP, BFP, YFP,13-galactosidase, chloramphenicol, acetyltransferase or luciferase. 95.A composition comprising the regulatory sequence as defined in claim 1,a recombinant expression molecule comprising a nucleotide sequence to beexpressed positioned under the control of such regulatory sequence, or avector or host cell comprising such a recombinant expression molecule.96. The composition of claim 95, which is a pharmaceutical composition,optionally comprising a pharmaceutically acceptable carrier or diluent.97. The composition of claim 95, which is a diagnostic composition,optionally further comprising suitable means of detection.
 98. A methodfor the detection of (a) proliferative neuronal determined cell(s)comprising the steps of: (a) expressing in a plurality of cells arecombinant nucleic acid molecule as defined in claim 58; and (b)selecting cells which express a heterologous nucleotide sequence underthe control of the regulatory sequence.
 99. The method of claim 98,which comprises in step (b) the detection of an expressed, heterologousmarker or reporter gene.
 100. The method of claim 42 which comprises thedetection of a fluorescent marker or reporter or an expressed enzyme.101. The method of claim 99, which comprises a FACS analysis, MACSanalysis or affinity isolation.
 102. The method of claim 99, furthercomprising separating the cell(s) which express(es) a heterologousnucleotide sequence under the control of the regulatory sequence from(a) cell(s) which do not express said heterologous nucleotide sequenceor which comprises an additional step of separating (a) cell(s) whichcomprise an activated regulatory sequence.
 103. A method for screeningof compounds capable of regulating neural stem cell activity,neurogenesis and/or neuronal differentiation comprising the steps of:(a) obtaining a compound that is to be tested for its ability toregulate neural stem cell activity, neurogenesis and/or neuronaldifferentiation; (b) contacting a DNA segment in accordance with claim58, or a cell comprising such a DNA segment, with said compound; and (c)detecting whether said compound(s) is/are capable of interacting withsaid regulatory sequence.
 104. A method for the preparation of apharmaceutical composition for the treatment of a neurological disorderor disease comprising the steps of the method of claim 103 whereby saidcompound capable of regulating neural stem cell activity, neurogenesisand/or neuronal differentiation obtained by said method is furtherformulated with a pharmaceutically acceptable carrier, excipients and/ordiluent.